Aminoalkyl glucosaminide phosphate compounds and their use as adjuvants and immunoeffectors

ABSTRACT

Aminoalkyl glucosaminide phosphate (AGP) compounds that are adjuvants and immunoeffectors are described and claimed. The compounds have a 2-deoxy-2-amino glucose in glycosidic linkage with an aminoalkyl (aglycon) group. Compounds are phosphorylated at the 4 or 6 carbon on the glucosaminide ring and comprise three 3-alkanoyloxyalkanoyl residues. The compounds augment antibody production in immunized animals as well as stimulate cytokine production and activate macrophages. Compositions and methods for using the compounds as adjuvants and immunoeffectors are also disclosed.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to aminoalkyl glucosaminidephosphate (AGP) compounds that have activity as adjuvants andimmunoeffectors, and to methods employing and compositions comprisingAGPs.

[0003] 2. Description of the Related Art

[0004] Humoral immunity and cell-mediated immunity are the two majorbranches of the mammalian immune response. Humoral immunity involves thegeneration of antibodies to foreign antigens. Antibodies are produced byB-lymphocytes. Cell-mediated immunity involves the activation ofT-lymphocytes that either act upon infected cells bearing foreignantigens or stimulate other cells to act upon infected cells. Bothbranches of the mammalian immune system are important in fightingdisease. Humoral immunity is the major line of defense against bacterialpathogens. In the case of viral disease, the induction of cytotoxic Tlymphocytes (CTLs) appears to be crucial for protective immunity. Aneffective vaccine stimulates both branches of the immune system toprotect against disease.

[0005] Vaccines present foreign antigens from disease causing agents toa host so that the host can mount a protective immune response. Oftenvaccine antigens are killed or attenuated forms of the microbes thatcause the disease. The presence of non-essential components and antigensin these killed or attenuated vaccines has encouraged considerableefforts to refine vaccine components including developing well-definedsynthetic antigens using chemical and recombinant techniques. Therefinement and simplification of microbial vaccines, however, has led toa concomitant loss in potency. Low-molecular weight synthetic antigens,though devoid of potentially harmful contaminants, are themselves notvery immunogenic. These observations have led investigators to addadjuvants to vaccine compositions to potentiate the activity of therefined vaccine components.

[0006] Presently, the only adjuvant licensed for human use in the UnitedStates is alum, a group of aluminum salts (e.g., aluminum hydroxide,aluminum phosphate) in which vaccine antigens are formulated.Particulate carriers like alum serve to promote the uptake, processingand presentation of soluble antigens by macrophages. Alum, however, isnot without side-effects and enhances humoral (antibody) immunity only.

[0007] An effective adjuvant potentiates both a humoral and cellularimmune response in vaccinated animals. Further, an adjuvant must enhancea host's natural immune response and not aggravate the host system. Awell-defined synthetic adjuvant free from extraneous matter, which isstable and easy to manufacture, would provide these qualities. Compoundsthat have been prepared and tested for adjuvanticity (Shimizu et al.1985, Bulusu et al. 1992, Ikeda et al. 1993, Shimizu et al. 1994,Shimizu et al. 1995, Miyajima et al. 1996), however, often display toxicproperties, are unstable and/or have unsubstantial immunostimulatoryeffects.

[0008] The discovery and development of effective adjuvants is essentialfor improving the efficacy and safety of existing vaccines. Adjuvantsimpart synthetic peptides and carbohydrate antigens with sufficientimmunogenicity to insure the success of the synthetic vaccine approach.There remains a need for new compounds having potent immunomodulatingeffects.

BRIEF SUMMARY OF THE INVENTION

[0009] The compounds of the subject invention are aminoalkylglucosaminide phosphate compounds (AGPs) that are adjuvants andimmunoeffectors. An aminoalkyl (aglycon) group is glycosidically linkedto a 2-deoxy-2-amino-α-D-glucopyranose (glucosaminide) to form the basicstructure of the claimed molecules. The compounds are phosphorylated atthe 4 or 6 carbon on the glucosaminide ring. Further, the compoundspossess three 3-alkanoyloxyalkanoyl residues.

[0010] The compounds of the subject invention are immunoeffectormolecules augmenting antibody production in immunized animals,stimulating cytokine production and activating macrophages. Inaccordance with the subject invention, methods for using these compoundsas adjuvants and immunoeffectors are disclosed.

[0011] According to the present invention, methods for inducing animmune response employ the administration of one or more AGP eitheralone or in conjunction with one or more antigen such as a proteinantigen or a polynucleotide that expresses a protein antigen. Inventivecompositions for inducing an immune response employ one or more AGEeither alone or in combination with one or more antigen such as aprotein antigen or a polynucleotide that expresses a protein antigen.Exemplary antigens include, but are not limited to, tumor antigens andinfectious disease antigens. Induction of an immune response may bedetermined by measuring antibody in immunized animals. Such measurementsmay include a determination of seroconversion and/or seroprotection.Alternatively, or additionally, an immune response may be determined bymeasureing the production of cytokines and/or the stimulation of acell-mediated immune response including a cytotoxic T-lymphocyteresponse.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 is a graph depicting the percentage of human subjectsachieving seroprotection following administration of Hepatitis B SurfaceAntigen (AgB) alone or in combination with the AGP designated RC-210-04(B19 in Table land Example 20 herein below; chemical name2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt). Plot symbols show the percentages of efficacyevaluable (EE) population subjects who achieved seroprotection(anti-HBsAg titer of ≧10 MIU/mL) at the Day 90 visit. The error barsshow 95% confidence intervals for the percentages of subjects achievingseroprotection.

[0013]FIG. 2 is a graph depicting the percentage of human subjectsachieving seroprotection (anti-HBsAg titer of ≧10 MIU/mL). Plot symbolsshow the percentages of EE population subjects who achievedseroprotection at each of the Day 30, 60, and 90 visits. The error barsshow 95% confidence intervals for the percentages of subjects achievingseroprotection.

[0014]FIG. 3 is a graph depicting the percentage of human subjectsachieving seroconversion (anti-HBsAg titer of ≧1 MIU/mL). Plot symbolsshow the percentages of EE population subjects who achievedseroconversion at each of the Day 30, 60, and 90 visits. The error barsshow 95% confidence intervals for the percentages of subjects achievingseroconversion.

[0015]FIG. 4 is a graph depicting the distribution of Anti-HBsAg Titersin human subjects. Each curve shows a nonparametric estimate of thedistribution of anti-HBsAg titers in a treatment group at a particularvisit. Dashed curves correspond to the AgB group, solid curvescorrespond to the AgB/RC-210-04 treatment group. The numbers next to thecurves show the nominal study day (visit) at which the data werecollected. The area under each curve is proportional to the observedfraction of patients for whom a non-zero titer was measured.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Aminoalkyl glucosaminide phosphate (AGP) Compounds

[0017] The compounds of the subject invention are adjuvant andimmunoeffector molecules that are aminoalkyl glucosaminide phosphates(AGPs). The compounds comprise a 2-deoxy-2-amino-α-D-glucopyranose(glucosaminide) in glycosidic linkage with an aminoalkyl (aglycon)group. Compounds are phosphorylated at the 4 or 6 carbon on theglucosaminide ring and have three alkanoyloxyalkanoyl residues. Thecompounds of the subject invention are described generally by Formula I,

[0018] wherein X represents an oxygen or sulfur atom in either the axialor equitorial position, Y represents an oxygen atom or NH group, “n”,“m”, “p” and “q” are integers from 0 to 6, R₁, R₂, and R₃ representnormal fatty acyl residues having 1 to 20 carbon atoms and where one ofR₁, R₂ or R₃ is optionally hydrogen, R₄ and R₅ are hydrogen or methyl,R₆ and R₇ are hydrogen, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo,sulfooxy, amino, mercapto, cyano, nitro, formyl or carboxy and estersand amides thereof; R₈ and R₉ are phosphono or hydrogen. Theconfiguration of the 3′ stereogenic centers to which the normal fattyacyl residues are attached is R or S, but preferably R. Thestereochemistry of the carbon atoms to which R₄ or R₅ are attached canbe R or S. All stereoisomers, both enantiomers and diastereomers, andmixtures thereof, are considered to fall within the scope of the subjectinvention.

[0019] The heteroatom X of the compounds of the subject invention can beoxygen or sulfur. In a preferred embodiment, X is oxygen and typicallyin the equitorial position. Although the stability of the moleculescould be effected by a substitution at X, the immunomodulating activityof molecules with these substitutions is not expected to change.

[0020] The number of carbon atoms between heteroatom X and the aglyconnitrogen atom is determined by variables “n” and “m”. Variables “n” and“m” can be integers from 0 to 6. In a preferred embodiment, the totalnumber of carbon atoms between heteroatom X and the aglycon nitrogenatom is from about 2 to about 6 and most preferably from about 2 toabout 4.

[0021] The compounds of the subject invention are aminoalkylglucosaminide compounds that are phosphorylated. Compounds can bephosphorylated at position 4 or 6 (R₈ or R₉) on the glucosaminide ringand are most effective if phosphorylated on at least one of thesepositions. In a preferred embodiment, R₈ is phosphono and R₉ ishydrogen.

[0022] In one embodiment, the compounds of the subject invention arehexaacylated; that is, they contain a total of six fatty acid residues.The aminoalkyl glucosaminide moiety is acylated at the 2-amino and3-hydroxyl groups of the glucosaminide unit and at the amino group ofthe aglycon unit with 3-hydroxyalkanoyl residues. In Formula I, thesethree positions are acylated with 3-hydroxytetradecanoyl moieties. The3-hydroxytetradecanoyl residues are, in turn, substituted with normalfatty acids (R₁-R₃), providing three 3-n-alkanoyloxytetradecanoylresidues or six fatty acid groups in total.

[0023] In another embodiment, the compounds of the subject invention arepentaacylated; that is, they contain a total of five fatty acidresidues. More specifically, the 3-hydroxytetradecanoyl residues ofFormula I are substituted with normal fatty acids at two of the threeR₁, R₂ and R₃ positions, with the third R₁, R₂ or R₃ position beinghydrogen. In other words, at least one of —OR₁, —OR₂ or —OR₃ ishydroxyl.

[0024] The chain length of normal fatty acids R₁-R₃ can be from 1 toabout 20, and typically from about 7 to about 16 carbons. Preferably,R₁-R₃ are from about 9 to about 14 carbons. The chain lengths of thesenormal fatty acids can be the same or different. Although, only normalfatty acids are described, it is expected that unsaturated fatty acids(i.e. fatty acid moieties having double or triple bonds) substituted atR₁-R₃ on the compounds of the subject invention would producebiologically active molecules. Further, slight modifications in thechain length of the 3-hydroxyalkanoyl residues are not expected todramatically effect biological activity.

[0025] The compounds of the subject invention are synthesized bycoupling an N-acyloxyacylated or N-protected aminoalkanol oraminoalkanethiol (aglycon unit) with a suitably protected and/or3-O-acyloxyacylated glucosaminide unit. In one preferred method forpreparing the compounds of the subject invention (Scheme 1), anN-(2,2,2-trichloroethoxycarbonyl (Troc))-protected glycosyl halide 1(Z=F, Cl, Br) is coupled with anN-[(R)-3-n-alkanoyloxytetradecanoyl]aminoalkanol or thiol 2 (possessingR₅ and R₆ in suitably protected form) via a Koenigs-Knorr type reactionin the presence of mercury or silver salts to give glycosideintermediate 3. Preferably, the glucosaminide unit 1 possesses ananomeric chloride atom (Z=Cl), and the coupling catalyst is silvertrifluoromethanesulfonate. Intermediate 3 can also be prepared bycoupling the aglycon unit 2 with an N-Troc-protected glycosyl acetate(Z=OAc) or related activated derivative in the presence of a Lewis acidsuch as boron trifluoride etherate. By “activated” is meant having anappropriate displaceable leaving group “Z” attached to the anomericcenter of the glucosaminide unit. Glucosaminide unit 1 bears an(R)-3-n-alkanoyloxytetradecanoyl residue on the 3-position, and suitableprotecting groups on the 6-hydroxyl and 4-phosphate moieties. Typicalprotecting groups for the phosphate group include, but are not limitedto, phenyl, benzyl, and o-xylyl. The phosphate group is protectedpreferably with two phenyl groups. The 6-position can be temporarilyprotected by blocking groups commonly used in sugar chemistry such assilyl, benzyl, or benzyloxymethyl ethers or, alternatively, an alkylcarbonate. The 6-hydroxyl group is protected preferably as a1,1-dimethyl-2,2,2-trichloroethyl carbonate (TCBOC).

[0026] The trichloroethyl-based protecting group(s) in the Koenigs-Knorrcoupled product 3 are removed with zinc and the glucosaminide nitrogenis selectively acylated with a (R)-3-n-alkanoyloxytetradecanoic acid 4in the presence of a suitable coupling reagent to give the hexaacylatedderivative 5. The remaining protecting groups in 5 are then cleaved bycatalytic hydrogenation in the presence of a palladium or platinumcatalyst or by other appropriate means to give compounds of Formula (I).

[0027] A suitable starting material for the synthesis of glycosyl donor1 is 2-(trimethylsilyl)ethyl²-amino-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside which can beprepared from commercially available D-glucosaminide hydrochloride usingpublished procedures. The conversion of the 2-(trimethylsilyl)ethylglycoside starting material to glycosyl donor 1 can be achieved bymethods known in the art or modifications thereof which are describedherein. The aglycon unit 2 can be prepared by N-acyloxyacylation ofcommercially available starting materials with an appropriate(R)-3-n-alkanoyloxytetradecanoic acid 4, or N-acyloxyacylation ofstarting materials that can be obtained by known methods in the chemicalliterature. Alternatively, the N-acyloxyacyl residue in 2 can besubstituted with an appropriate amine protecting group which is removedsubsequent to the coupling reaction such as is described in the secondpreferred embodiment below.

[0028] In a second preferred method for preparing the compounds of thesubject invention (Scheme 2), introduction of the(R)-3-n-alkanoyloxytetradecanoyl and phosphate groups into theglucosaminide and aglycon units is performed subsequent to theglycosylation (coupling) reaction using N- and O-protecting groupssuitable for the chemical differentiation of the amino and hydroxylgroups present. Preferably, the N-Troc-protected glycosyl donor 6 iscoupled with an N-allyloxycarbonyl (AOC)-protected aminoalkanol or thiol7 in the presence of an appropriate catalyst to give the aminoalkylβ-glycoside 8. Most preferably, the glycosyl donor 6 possesses ananomeric acetoxy group (Z=OAc), and the coupling catalyst is borontrifluoride etherate. Other N-protecting groups for the aglycon aminogroup include, but are not limited to, commonly employed carbamatesobvious to one skilled in the art such as t-butyl (t-BOC), benzyl (Cbz),2,2,2-trichloroethyl (Troc), and 9-fluorenylmethyl(Fmoc).

[0029] Base-induced cleavage of the acetate groups in coupling product 8and 4,6-acetonide formation under standard conditions known in the artgives intermediate 9.3-O-Acylation of 9 with(R)-3-n-alkanoyloxytetradecanoic acid 4, followed bypalladium(0)-mediated removal of the aglycon N-AOC group and N-acylationwith (R)-3-n-alkanoyloxytetradecanoic acid 4 provides intermediate 10.Acetonide hydrolysis and functionalization of the 4- and 6-positions asdescribed herein for the preparation of glycosyl donor 1 givesintermediate 3 (Y=O), which is then processed as in Scheme 1 to affordcompounds of general Formula (I).

[0030] AGP-Based Compositions

[0031] In compositions for eliciting an immune response, the AGPs of thesubject invention are administered to a warm-blooded animal, includinghumans, with an antigen such as a protein or polypeptide antigen or apolynucleotide that expresses a protein or polypeptide antigen. Theamount of antigen administered to elicit a desired response can bereadily determined by one skilled in the art and will vary with the typeof antigen administered, route of administration and immunizationschedule. For example, 0.2 μg of tetanus toxoid administered with theclaimed compounds subcutaneously to a mouse in two immunization 21 daysapart elicited a humoral immune response to that antigen.

[0032] The AGPs of the subject invention, as well as monophosphoryllipid A (MPL®), provide immediate protection via a non-specificresistance effect, see Persing et al., WIPO Publication WO 01/90129,Nov. 29, 2001. As described herein, AGPs of the subject inventionadministered with an antigen lead to an acquired mucosal immune responsewithin three to four weeks. Weekly administration of such compounds, viaan intranasal route for example, over a four-week period would providerapid and durable protection by combining the protection provided by theinitial innate immune response, followed by the acquired immune responseto the antigen of interest.

[0033] AGP-Based Compositions Comprising One or More Polypeptide

[0034] As used herein, the term “polypeptide” is used in itsconventional meaning, i.e., as a sequence of amino acids. Thepolypeptides are not limited to a specific length of the product; thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

[0035] The polypeptides of the present invention are sometimes hereinreferred to as tumor proteins or tumor polypeptides, as an indicationthat their identification has been based at least in part upon theirincreased levels of expression in tumor samples. Thus, a “tumorpolypeptide” or “tumor protein,” refers generally to a polypeptidesequence of the present invention, or a polynucleotide sequence encodingsuch a polypeptide, that is expressed in a substantial proportion oftumor samples, for example preferably greater than about 20%, morepreferably greater than about 30%, and most preferably greater thanabout 50% or more of tumor samples tested, at a level that is at leasttwo fold, and preferably at least five fold, greater than the level ofexpression in normal tissues, as determined using a representative assayprovided herein.

[0036] In certain preferred embodiments, the polypeptides of theinvention are immunogenic, i.e., they react detectably within animmunoassay (such as an ELISA or T-cell stimulation assay) with antiseraand/or T-cells from a patient with cancer. Screening for immunogenicactivity can be performed using techniques well known to the skilledartisan. For example, such screens can be performed using methods suchas those described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

[0037] As would be recognized by the skilled artisan, immunogenicportions of the polypeptides disclosed herein are also encompassed bythe present invention. An “immunogenic portion,” as used herein, is afragment of an immunogenic polypeptide of the invention that itself isimmunologically reactive (i.e., specifically binds) with the B-cellsand/or T-cell surface antigen receptors that recognize the polypeptide.Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Suchtechniques include screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

[0038] In one preferred embodiment, an immunogenic portion of apolypeptide of the present invention is a portion that reacts withantisera and/or T-cells at a level that is not substantially less thanthe reactivity of the full-length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Preferably, the level of immunogenic activityof the immunogenic portion is at least about 50%, preferably at leastabout 70% and most preferably greater than about 90% of theimmunogenicity for the full-length polypeptide. In some instances,preferred immunogenic portions will be identified that have a level ofimmunogenic activity greater than that of the corresponding full-lengthpolypeptide, e.g., having greater than about 100% or 150% or moreimmunogenic activity.

[0039] In certain other embodiments, illustrative immunogenic portionsmay include peptides in which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

[0040] In another embodiment, a polypeptide composition of the inventionmay also comprise one or more polypeptides that are immunologicallyreactive with T cells and/or antibodies generated against a polypeptideof the invention, particularly a polypeptide having an amino acidsequence disclosed herein, or to an immunogenic fragment or variantthereof.

[0041] In another embodiment of the invention, polypeptides are providedthat comprise one or more polypeptides that are capable of eliciting Tcells and/or antibodies that are immunologically reactive with one ormore polypeptides described herein, or one or more polypeptides encodedby contiguous nucleic acid sequences contained in the polynucleotidesdisclosed herein, or immunogenic fragments or variants thereof.

[0042] Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

[0043] Within other illustrative embodiments, a polypeptide may be afusion polypeptide that comprises multiple polypeptides as describedherein, or that comprises at least one polypeptide as described hereinand an unrelated sequence, such as a known tumor protein. A fusionpartner may, for example, assist in providing T helper epitopes (animmunological fusion partner), preferably T helper epitopes recognizedby humans, or may assist in expressing the protein (an expressionenhancer) at higher yields than the native recombinant protein. Certainpreferred fusion partners are both immunological and expressionenhancing fusion partners. Other fusion partners may be selected so asto increase the solubility of the polypeptide or to enable thepolypeptide to be targeted to desired intracellular compartments. Stillfurther fusion partners include affinity tags, which facilitatepurification of the polypeptide.

[0044] Fusion polypeptides may generally be prepared using standardtechniques, including chemical conjugation. Preferably, a fusionpolypeptide is expressed as a recombinant polypeptide, allowing theproduction of increased levels, relative to a non-fused polypeptide, inan expression system. Briefly, DNA sequences encoding the polypeptidecomponents may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion polypeptide that retains the biologicalactivity of both component polypeptides.

[0045] A peptide linker sequence may be employed to separate the firstand second polypeptide components by a distance sufficient to ensurethat each polypeptide folds into its secondary and tertiary structures.Such a peptide linker sequence is incorporated into the fusionpolypeptide using standard techniques well known in the art. Suitablepeptide linker sequences may be chosen based on the following factors:(1) their ability to adopt a flexible extended conformation; (2) theirinability to adopt a secondary structure that could interact withfunctional epitopes on the first and second polypeptides; and (3) thelack of hydrophobic or charged residues that might react with thepolypeptide functional epitopes. Preferred peptide linker sequencescontain Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala may also be used in the linker sequence. Amino acidsequences which may be usefully employed as linkers include thosedisclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc.Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 andU.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 toabout 50 amino acids in length. Linker sequences are not required whenthe first and second polypeptides have non-essential N-terminal aminoacid regions that can be used to separate the functional domains andprevent steric interference.

[0046] The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

[0047] The fusion polypeptide can comprise a polypeptide as describedherein together with an unrelated immunogenic protein, such as animmunogenic protein capable of eliciting a recall response. Examples ofsuch proteins include tetanus, tuberculosis and hepatitis proteins (see,for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

[0048] In one preferred embodiment, the immunological fusion partner isderived from a Mycobacterium sp., such as a Mycobacteriumtuberculosis-derived Ra12 fragment. Ra12 compositions and methods fortheir use in enhancing the expression and/or immunogenicity ofheterologous polynucleotide/polypeptide sequences is described in U.S.Patent Application No. 60/158,585, the disclosure of which isincorporated herein by reference in its entirety. Briefly, Ra12 refersto a polynucleotide region that is a subsequence of a Mycobacteriumtuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 KDmolecular weight encoded by a gene in virulent and avirulent strains ofM. tuberculosis. The nucleotide sequence and amino acid sequence ofMTB32A have been described (for example, U.S. Patent Application No.60/158,585; see also, Skeiky et al., Infection and Immun. (1999)67:3998-4007, incorporated herein by reference). C-terminal fragments ofthe MTB32A coding sequence express at high levels and remain as asoluble polypeptides throughout the purification process. Moreover, Ra12may enhance the immunogenicity of heterologous immunogenic polypeptideswith which it is fused. One preferred Ra12 fusion polypeptide comprisesa 14 KD C-terminal fragment corresponding to amino acid residues 192 to323 of MTB32A. Other preferred Ra12 polynucleotides generally compriseat least about 15 consecutive nucleotides, at least about 30nucleotides, at least about 60 nucleotides, at least about 100nucleotides, at least about 200 nucleotides, or at least about 300nucleotides that encode a portion of a Ra12 polypeptide. Ra12polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Ra12 polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Ra12 polynucleotide variants maycontain one or more substitutions, additions, deletions and/orinsertions such that the biological activity of the encoded fusionpolypeptide is not substantially diminished, relative to a fusionpolypeptide comprising a native Ra12 polypeptide. Variants preferablyexhibit at least about 70% identity, more preferably at least about 80%identity and most preferably at least about 90% identity to apolynucleotide sequence that encodes a native Ra12 polypeptide or aportion thereof.

[0049] Within other preferred embodiments, an immunological fusionpartner is derived from protein D, a surface protein of thegram-negative bacterium Haemophilus influenza B (WO 91/18926).Preferably, a protein D derivative comprises approximately the firstthird of the protein (e.g., the first N-terminal 100-110 amino acids),and a protein D derivative may be lipidated. Within certain preferredembodiments, the first 109 residues of a Lipoprotein D fusion partner isincluded on the N-terminus to provide the polypeptide with additionalexogenous T-cell epitopes and to increase the expression level in E.coli (thus functioning as an expression enhancer). The lipid tailensures optimal presentation of the antigen to antigen presenting cells.Other fusion partners include the non-structural protein from influenzaevirus, NS 1 (hemaglutinin). Typically, the N-terminal 81 amino acids areused, although different fragments that include T-helper epitopes may beused.

[0050] In another embodiment, the immunological fusion partner is theprotein known as LYTA, or a portion thereof (preferably a C-terminalportion). LYTA is derived from Streptococcus pneumoniae, whichsynthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encodedby the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin thatspecifically degrades certain bonds in the peptidoglycan backbone. TheC-terminal domain of the LYTA protein is responsible for the affinity tothe choline or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798,1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

[0051] Yet another illustrative embodiment involves fusion polypeptides,and the polynucleotides encoding them, wherein the fusion partnercomprises a targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

[0052] Polypeptides of the invention are prepared using any of a varietyof well known synthetic and/or recombinant techniques. Polypeptides,portions and other variants generally less than about 150 amino acidscan be generated by synthetic means, using techniques well known tothose of ordinary skill in the art. In one illustrative example, suchpolypeptides are synthesized using any of the commercially availablesolid-phase techniques, such as the Merrifield solid-phase synthesismethod, where amino acids are sequentially added to a growing amino acidchain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipmentfor automated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems Division (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions.

[0053] In general, polypeptide compositions (including fusionpolypeptides) of the invention are isolated. An “isolated” polypeptideis one that is removed from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

[0054] AGP-based Compositions Comprising One or More Polynucleotide

[0055] The present invention, in other aspects, provides AGP-basedcompositions comprising one or more polynucleotide that encodes apolypeptide antigen as set forth herein above. The terms “DNA” and“polynucleotide” are used essentially interchangeably herein to refer toa DNA molecule that has been isolated free of total genomic DNA of aparticular species. “Isolated,” as used herein, means that apolynucleotide is substantially away from other coding sequences, andthat the DNA molecule does not contain large portions of unrelatedcoding DNA, such as large chromosomal fragments or other functionalgenes or polypeptide coding regions. Of course, this refers to the DNAmolecule as originally isolated, and does not exclude genes or codingregions later added to the segment by the hand of man.

[0056] Polynucleotides may comprise a native sequence (i.e., anendogenous sequence that encodes a polypeptide/protein of the inventionor a portion thereof) or may comprise a sequence that encodes a variantor derivative, preferably and immunogenic variant or derivative, of sucha sequence. Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompass homologous genesof xenogenic origin.

[0057] In certain preferred embodiments, the polynucleotides describedabove, e.g., polynucleotide variants, fragments and hybridizingsequences, encode polypeptides that are immunologically cross-reactivewith an antigenic or immunogenic polypeptide as set forth herein above.In other preferred embodiments, such polynucleotides encode polypeptidesthat have a level of immunogenic activity of at least about 50%,preferably at least about 70%, and more preferably at least about 90% ofthat for a polypeptide sequence specifically set forth herein.

[0058] The polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol. For example, illustrativepolynucleotide segments with total lengths of about 10,000, about 5000,about 3000, about 2,000, about 1,000, about 500, about 200, about 100,about 50 base pairs in length, and the like, (including all intermediatelengths) are contemplated to be useful in many implementations of thisinvention.

[0059] Polynucleotides compositions of the present invention may beidentified, prepared and/or manipulated using any of a variety of wellestablished techniques (see generally, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y., 1989, and other like references). For example, apolynucleotide may be identified, as described in more detail below, byscreening a microarray of cDNAs for tumor-associated expression (i.e.,expression that is at least two fold greater in a tumor than in normaltissue, as determined using a representative assay provided herein).Such screens may be performed, for example, using the microarraytechnology of Affymetrix, Inc. (Santa Clara, Calif.) according to themanufacturer's instructions (and essentially as described by Schena etal., Proc. Natl. Acad. Sci. USA 93:10614-10619, 1996 and Heller et al.,Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997). Alternatively,polynucleotides may be amplified from cDNA prepared from cellsexpressing the proteins described herein, such as tumor cells.

[0060] Many template dependent processes are available to amplify atarget sequence of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g, Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

[0061] Any of a number of other template dependent processes, many ofwhich are variations of the PCR™ amplification technique, are readilyknown and available in the art. Illustratively, some such methodsinclude the ligase chain reaction (referred to as LCR), described, forexample, in Eur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No.4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.PCT/US87/00880; Strand Displacement Amplification (SDA) and Repair ChainReaction (RCR). Still other amplification methods are described in GreatBritain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well known to those of skill in the art.

[0062] An amplified portion of a polynucleotide of the present inventionmay be used to isolate a full length gene from a suitable library (e.g.,a tumor cDNA library) using well known techniques. Within suchtechniques, a library (cDNA or genomic) is screened using one or morepolynucleotide probes or primers suitable for amplification. Preferably,a library is size-selected to include larger molecules. Random primedlibraries may also be preferred for identifying 5′ and upstream regionsof genes. Genomic libraries are preferred for obtaining introns andextending 5′ sequences.

[0063] For hybridization techniques, a partial sequence may be labeled(e.g., by nick-translation or end-labeling with ³²P) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full-length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

[0064] Alternatively, amplification techniques, such as those describedabove, can be useful for obtaining a full length coding sequence from apartial cDNA sequence. One such amplification technique is inverse PCR(see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which usesrestriction enzymes to generate a fragment in the known region of thegene. The fragment is then circularized by intramolecular ligation andused as a template for PCR with divergent primers derived from the knownregion. Within an alternative approach, sequences adjacent to a partialsequence may be retrieved by amplification with a primer to a linkersequence and a primer specific to a known region. The amplifiedsequences are typically subjected to a second round of amplificationwith the same linker primer and a second primer specific to the knownregion. A variation on this procedure, which employs two primers thatinitiate extension in opposite directions from the known sequence, isdescribed in WO 96/38591. Another such technique is known as “rapidamplification of cDNA ends” or RACE. This technique involves the use ofan internal primer and an external primer, which hybridizes to a polyAregion or vector sequence, to identify sequences that are 5′ and 3′ of aknown sequence. Additional techniques include capture PCR (Lagerstrom etal., PCR Methods Applic. 1:111-19,1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60,1991). Other methods employingamplification may also be employed to obtain a full-length cDNAsequence.

[0065] In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well-known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full-length DNA sequences may also beobtained by analysis of genomic fragments.

[0066] In other embodiments of the invention, polynucleotide sequencesor fragments thereof which encode polypeptides set forth herein above,or fusion proteins or functional equivalents thereof, may be used inrecombinant DNA molecules to direct expression of a polypeptide inappropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide.

[0067] Sequences encoding a desired polypeptide may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).

[0068] In order to express a desired polypeptide, the nucleotidesequences encoding the polypeptide, or functional equivalents, may beinserted into appropriate expression vector, i.e., a vector thatcontains the necessary elements for the transcription and translation ofthe inserted coding sequence. Methods well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

[0069] The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used.

[0070] In mammalian cells, a number of viral-based expression systemsare generally available. For example, in cases where an adenovirus isused as an expression vector, sequences encoding a polypeptide ofinterest may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus that is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

[0071] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding a polypeptide of interest.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

[0072] A variety of protocols for detecting and measuring the expressionof polynucleotide-encoded products, using either polyclonal ormonoclonal antibodies specific for the product are known in the art.Examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on a given polypeptide may bepreferred for some applications, but a competitive binding assay mayalso be employed. These and other assays are described, among otherplaces, in Hampton, R. et al. (1990; Serological Methods, a LaboratoryManual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J.Exp. Med. 158:1211-1216).

[0073] AGP-Based Pharmaceutical Compositions

[0074] The AGP compounds of the subject invention are adjuvants andimmunoeffectors which enhance the generation of antibody in immunizedanimals, stimulate the production of cytokines and stimulate acell-mediated immune response including a cytotoxic T-lymphocyteresponse. In methods for effecting the immune response of an individual,the compounds and compositions of the subject invention can beformulated with a pharmaceutically acceptable carrier for injection oringestion. As used herein, “pharmaceutically acceptable carrier” means amedium that does not interfere with the immunomodulatory activity of theactive ingredient and is not toxic to the patient to whom it isadministered. Pharmaceutically acceptable carriers include oil-in-wateror water-in-oil emulsions, aqueous compositions, liposomes, microbeadsand microsomes. For example, the carrier may be a microsphere ormicroparticle having a compound of this invention within the matrix ofthe sphere or particle or adsorbed on the surface of the sphere orparticle. The carrier may also be an aqueous solution or micellardispersion containing triethylamine, triethanolamine or other agent thatrenders the formulation alkaline in nature, or a suspension containingaluminum hydroxide, calcium hydroxide, calcium phosphate or tyrosineadsorbate.

[0075] Formulations of the compounds of the subject invention that canbe administered parenterally, i.e. intraperitoneally, subcutaneously orintramuscularly include the following preferred carriers. Examples ofpreferred carriers for subcutaneous use include a phosphate bufferedsaline (PBS) solution and 0.01-0.1% triethanolamine in USP Water forInjection. Suitable carriers for intramuscular injection include 10% USPethanol, 40% propylene glycol and the balance an acceptable isotonicsolution such as 5% dextrose.

[0076] Examples of preferred carriers for intravenous use include 10%USP ethanol, 40% USP propylene glycol and the balance USP Water forInjection. Another acceptable carrier includes 10% USP ethanol and USPWater for Injection; yet another acceptable carrier is 0.01-0.1%triethanolamine in USP Water for Injection. Pharmaceutically acceptableparenteral solvents are such as to provide a solution or dispersion maybe filtered through a 5 micron filter without removing the activeingredient.

[0077] Examples of carriers for administration via mucosal surfacesdepend upon the particular route. When administered orally,pharmaceutical grades of mannitol, starch, lactose, magnesium stearate,sodium saccharide, cellulose, magnesium carbonate and the like, withmannitol being preferred. When administered intranasally, polyethyleneglycol or glycols, sucrose, and/or methylcellulose, and preservativessuch as benzalkonium chloride, EDTA, may be used, with polyethyleneglycols being preferred, and when administered by inhalation, suitablecarriers are polyethylene glycol or glycols, methylcellulose, dispensingagents, and preservatives, with polyethylene glycols being preferred.

[0078] While any suitable carrier known to those of ordinary skill inthe art may be employed in the pharmaceutical compositions of thisinvention, the type of carrier will typically vary depending on the modeof administration. Compositions of the present invention may beformulated for any appropriate manner of administration, including forexample, topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

[0079] Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

[0080] The compounds of the subject invention are administered to anindividual in “an effective amount” to effect or enhance theindividual's immune response. As used herein, “an effective amount” isthat amount which shows a response over and above the vehicle ornegative controls. The precise dosage of the compounds of the subjectinvention to be administered to a patient will depend upon theparticular AGP used, the route of administration, the pharmaceuticalcomposition, and the patient. For example, when administeredsubcutaneously to enhance an antibody response, the amount of AGP usedis from 1 to about 250 micrograms, preferably from about 25 to about 50micrograms based upon administration to a typical 70 kg adult patient.

[0081] In another embodiment, illustrative immunogenic compositions,e.g., immunogenic and/or vaccine compositions, of the present inventioncomprise DNA encoding one or more of the polypeptides as describedabove, such that the polypeptide is generated in situ. As noted above,the polynucleotide may be administered within any of a variety ofdelivery systems known to those of ordinary skill in the art. Indeed,numerous gene delivery techniques are well known in the art, such asthose described by Rolland, Crit. Rev. Therap. Drug Carrier Systems15:143-198, 1998, and references cited therein. Appropriatepolynucleotide expression systems will, of course, contain the necessaryregulatory DNA regulatory sequences for expression in a patient (such asa suitable promoter and terminating signal).

[0082] Therefore, in certain embodiments, polynucleotides encodingimmunogenic polypeptides described herein are introduced into suitablemammalian host cells for expression using any of a number of knownviral-based systems. In one illustrative embodiment, retrovirusesprovide a convenient and effective platform for gene delivery systems. Aselected nucleotide sequence encoding a polypeptide of the presentinvention can be inserted into a vector and packaged in retroviralparticles using techniques known in the art. The recombinant virus canthen be isolated and delivered to a subject. A number of illustrativeretroviral systems have been described (e.g., U.S. Pat. No. 5,219,740;Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990)Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; andBoris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0083] In addition, a number of illustrative adenovirus-based systemshave also been described. Unlike retroviruses which integrate into thehost genome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

[0084] Various adeno-associated virus (AAV) vector systems have alsobeen developed for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

[0085] Additional viral vectors useful for delivering thepolynucleotides encoding polypeptides of the present invention by genetransfer include those derived from the pox family of viruses, such asvaccinia virus and avian poxyirus. By way of example, vaccinia virusrecombinants expressing the novel molecules can be constructed asfollows. The DNA encoding a polypeptide is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells that aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the polypeptideof interest into the viral genome. The resulting TK.sup.(−) recombinantcan be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

[0086] A vaccinia-based infection/transfection system can beconveniently used to provide for inducible, transient expression orcoexpression of one or more polypeptides described herein in host cellsof an organism. In this particular system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide orpolynucleotides of interest, driven by a T7 promoter. The polymeraseexpressed in the cytoplasm from the vaccinia virus recombinanttranscribes the transfected DNA into RNA that is then translated intopolypeptide by the host translational machinery. The method provides forhigh level, transient, cytoplasmic production of large quantities of RNAand its translation products. See, e.g., Elroy-Stein and Moss, Proc.Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl.Acad. Sci. USA (1986) 83:8122-8126.

[0087] Alternatively, avipoxyiruses, such as the fowlpox and canarypoxviruses, can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxyiruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[0088] Any of a number of alphavirus vectors can also be used fordelivery of polynucleotide compositions of the present invention, suchas those vectors described in U.S. Pat. Nos. 5,843,723; 6,015,686;6,008,035 and 6,015,694. Certain vectors based on Venezuelan EquineEncephalitis (VEE) can also be used, illustrative examples of which canbe found in U.S. Pat. Nos. 5,505,947 and 5,643,576.

[0089] Moreover, molecular conjugate vectors, such as the adenoviruschimeric vectors described in Michael et al. J. Biol. Chem. (1993)268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992)89:6099-6103, can also be used for gene delivery under the invention.

[0090] Additional illustrative information on these and other knownviral-based delivery systems can be found, for example, in Fisher-Hochet al., Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al.,Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,1990; U.S. Pat. Nos. 4,603,112,4,769,330, and 5,017,487; WO 89/01973;U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219,1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502,1993; Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al.,Cir. Res. 73:1202-1207, 1993.

[0091] In certain embodiments, a polynucleotide may be integrated intothe genome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

[0092] In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

[0093] In still another embodiment, a composition of the presentinvention can be delivered via a particle bombardment approach, many ofwhich have been described. In one illustrative example, gas-drivenparticle acceleration can be achieved with devices such as thosemanufactured by Powderject Pharmaceuticals PLC (Oxford, UK) andPowderject Vaccines Inc. (Madison, Wis.), some examples of which aredescribed in U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807;and EP Patent No. 0500 799. This approach offers a needle-free deliveryapproach wherein a dry powder formulation of microscopic particles, suchas polynucleotide or polypeptide particles, are accelerated to highspeed within a helium gas jet generated by a hand held device,propelling the particles into a target tissue of interest.

[0094] In a related embodiment, other devices and methods that may beuseful for gas-driven needle-less injection of compositions of thepresent invention include those provided by Bioject, Inc. (Portland,Oreg.), some examples of which are described in U.S. Pat. Nos.4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and5,993,412.

[0095] Within certain embodiments of the invention, the AGB-basedpharmaceutical composition is preferably one that induces an immuneresponse predominantly of the Th1 type. High levels of Th 1-typecytokines (e.g, IFN-γ, TNFα, IL-2 and IL-12) tend to favor the inductionof cell-mediated immune responses to an administered antigen. Incontrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 andIL-10) tend to favor the induction of humoral immune responses.Following application of an immunogenic composition as provided herein,a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

[0096] The pharmaceutical compositions of the invention will oftenfurther comprise one or more buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, bacteriostats, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes thatrender the formulation isotonic, hypotonic or weakly hypertonic with theblood of a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

[0097] The pharmaceutical compositions described herein may be presentedin unit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

[0098] The development of suitable dosing and treatment regimens forusing the particular compositions described herein in a variety oftreatment regimens, including e.g., oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art, some of which are briefly discussed below for generalpurposes of illustration.

[0099] In certain applications, the pharmaceutical compositionsdisclosed herein may be delivered via oral administration to an animal.As such, these compositions may be formulated with an inert diluent orwith an assimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

[0100] The active compounds may even be incorporated with excipients andused in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like (see, forexample, Mathiowitz et al., Nature 1997 Mar 27;386(6623):410-4; Hwang etal., Crit Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

[0101] Typically, these formulations will contain at least about 0.1% ofthe active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

[0102] For oral administration the compositions of the present inventionmay alternatively be incorporated with one or more excipients in theform of a mouthwash, dentifrice, buccal tablet, oral spray, orsublingual orally-administered formulation. Alternatively, the activeingredient may be incorporated into an oral solution such as onecontaining sodium borate, glycerin and potassium bicarbonate, ordispersed in a dentifrice, or added in a therapeutically-effectiveamount to a composition that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants. Alternatively thecompositions may be fashioned into a tablet or solution form that may beplaced under the tongue or otherwise dissolved in the mouth.

[0103] In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

[0104] Illustrative pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0105] In one embodiment, for parenteral administration in an aqueoussolution, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. In this connection, a sterile aqueous medium that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. Moreover, for humanadministration, preparations will of course preferably meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

[0106] In another embodiment of the invention, the compositionsdisclosed herein may be formulated in a neutral or salt form.Illustrative pharmaceutically-acceptable salts include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective.

[0107] The carriers can further comprise any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. The phrase“pharmaceutically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human.

[0108] In certain embodiments, the pharmaceutical compositions may bedelivered by intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering genes, nucleic acids, andpeptide compositions directly to the lungs via nasal aerosol sprays hasbeen described, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No.5,804,212. Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., J Controlled Release 1998 Mar2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U.S. Pat. No.5,725,871) are also well-known in the pharmaceutical arts. Likewise,illustrative transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045.

[0109] In certain embodiments, liposomes, nanocapsules, microparticles,lipid particles, vesicles, and the like, are used for the introductionof the compositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

[0110] The formation and use of liposome and liposome-like preparationsas potential drug carriers is generally known to those of skill in theart (see for example, Lasic, Trends Biotechnol 1998 Jul; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

[0111] Liposomes have been used successfully with a number of cell typesthat are normally difficult to transfect by other procedures, includingT cell suspensions, primary hepatocyte cultures and PC 12 cells(Renneisen et al., J. Biol. Chem. 1990 Sep 25;265(27):16337-42; Mulleret al., DNA Cell Biol. 1990 April; 9(3):221-9). In addition, liposomesare free of the DNA length constraints that are typical of viral-baseddelivery systems. Liposomes have been used effectively to introducegenes, various drugs, radiotherapeutic agents, enzymes, viruses,transcription factors, allosteric effectors and the like, into a varietyof cultured cell lines and animals. Furthermore, he use of liposomesdoes not appear to be associated with autoimmune responses orunacceptable toxicity after systemic delivery.

[0112] In certain embodiments, liposomes are formed from phospholipidsthat are dispersed in an aqueous medium and spontaneously formmultilamellar concentric bilayer vesicles (also termed multilamellarvesicles (MLVs).

[0113] Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March; 45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan 2;50(1-3):31-40;and U.S. Pat. No. 5,145,684.

[0114] Cancer Therapies

[0115] Immunologic approaches to cancer therapy are based on therecognition that cancer cells can often evade the body's defensesagainst aberrant or foreign cells and molecules, and that these defensesmight be therapeutically stimulated to regain the lost ground, e.g pgs.623-648 in Klein, Immunology (Wiley-Interscience, New York, 1982).Numerous recent observations that various immune effectors can directlyor indirectly inhibit growth of tumors has led to renewed interest inthis approach to cancer therapy, e.g. Jager, et al., Oncology2001;60(1):1-7; Renner, et al., Ann Hematol 2000 December; 79(12):651-9.

[0116] Four-basic cell types whose function has been associated withantitumor cell immunity and the elimination of tumor cells from the bodyare: i) B-lymphocytes which secrete immunoglobulins into the bloodplasma for identifying and labeling the nonself invader cells; ii)monocytes which secrete the complement proteins that are responsible forlysing and processing the immunoglobulin-coated target invader cells;iii) natural killer lymphocytes having two mechanisms for thedestruction of tumor cells, antibody-dependent cellular cytotoxicity andnatural killing; and iv) T-lymphocytes possessing antigen-specificreceptors and having the capacity to recognize a tumor cell carryingcomplementary marker molecules (Schreiber, H., 1989, in FundamentalImmunology (ed). W. E. Paul, pp. 923-955).

[0117] Cancer immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both. Moreover, it is wellestablished that induction of CD4⁺ T helper cells is necessary in orderto secondarily induce either antibodies or cytotoxic CD8⁺ T cells.Polypeptide antigens that are selective or ideally specific for cancercells offer a powerful approach for inducing immune responses againstcancer, and are an important aspect of the present invention.

[0118] Therefore, in further aspects of the present invention, thepharmaceutical compositions described herein may be used to stimulate animmune response against cancer. Within such methods, the pharmaceuticalcompositions described herein are administered to a patient, typically awarm-blooded animal, preferably a human. A patient may or may not beafflicted with cancer. Pharmaceutical compositions and vaccines may beadministered either prior to or following surgical removal of primarytumors and/or treatment such as administration of radiotherapy orconventional chemotherapeutic drugs. As discussed above, administrationof the pharmaceutical compositions may be by any suitable method,including administration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

[0119] Within certain embodiments, immunotherapy may be activeimmunotherapy, in which treatment relies on the in vivo stimulation ofthe endogenous host immune system to react against tumors with theadministration of immune response-modifying agents (such as polypeptidesand polynucleotides as provided herein).

[0120] Routes and frequency of administration of the therapeuticcompositions described herein, as well as dosage, will vary fromindividual to individual, and may be readily established using standardtechniques. In general, the pharmaceutical compositions and vaccines maybe administered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patients ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

[0121] In general, an appropriate dosage and treatment regimen providesthe active compound(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit. Such a response can be monitored byestablishing an improved clinical outcome (e.g., more frequentremissions, complete or partial, or longer disease-free survival) intreated patients as compared to non-treated patients. Increases inpreexisting immune responses to a tumor protein generally correlate withan improved clinical outcome. Such immune responses may generally beevaluated using standard proliferation, cytotoxicity or cytokine assays,which may be performed using samples obtained from a patient before andafter treatment.

[0122] The present invention is further described by way of thefollowing non-limiting Examples and Test Examples that are given forillustrative purposes only. It is important to note that theintroduction of the (R)-3-n-alkanoyloxytetradecanoyl groups and thephosphate group(s) into the glucosaminide and aglycon units do notnecessarily have to be performed in the order shown in Schemes 1 and 2or described in the Examples shown below.

[0123] Examples 1-43 describe methods of making the AGP compounds of thesubject invention. Test Examples 1-13 describe assays conducted to thedetermine the immunogenicity of these compounds. Test Example 14describes results of a human clinical study in which the AGP RC-210-04(designated B19 in Table 2; chemical name2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt) was co-administered with the Hepatits B SurfaceAntigen (AgB). Table 1 lists the chemical composition and experimentalreference numbers for each compound in these examples. TABLE 1 Exam-Ref. ple No. R₁-R₃ N p R₆ q R₇ 1 — — — — — — — 2 B1* n-C₁₃H₂₇CO 0 1 OH 0H 3 B2** n-C₁₃H₂₇CO 0 1 OH 0 H 4 B3 n-C₁₁H₂₃CO 0 1 OH 0 H 5 B4n-C₁₀H₂₁CO 0 1 OH 0 H 6 B5 n-C₉H₁₉CO 0 1 OH 0 H 7 B6*** n-C₉H₁₉CO 0 1 OH0 H 8 B7 n-C₈H₁₇CO 0 1 OH 0 H 9 B8 n-C₆H₁₃CO 0 1 OH 0 H 10 B9 n-C₉H₁₉CO1 1 OH 0 H 11 B10 n-C₉H₁₉CO 0 2 OH 0 H 12 B11 n-C₁₃H₂₇CO 0 0 CO₂H 0 H 13B12 n-C₁₁H₂₃CO 0 0 CO₂H 0 H 14 B13 n-C₁₀H₂₁CO 0 0 CO₂H 0 H 15 B14**n-C₉H₁₉CO 0 0 CO₂H 0 H 16 B15* n-C₉H₁₉CO 0 0 CO₂H 0 H 17 B16 n-C₈H₁₇CO 00 CO₂H 0 H 18 B17 n-C₇H₁₅CO 0 0 CO₂H 0 H 19 B18 n-C₆H₁₃CO 0 0 CO₂H 0 H20 B19 n-C₁₃H₂₇CO 0 0 H 0 H 21 B20 n-C₉H₁₉CO 0 0 H 0 H 22 B21 n-C₁₃H₂₇CO1 0 H 0 H 23 B22 n-C₁₃H₂₇CO 2 0 H 0 H 24 B23 n-C₁₃H₂₇CO 4 0 H 0 H 25 B24n-C₁₃H₂₇CO 0 0 CONH₂ 0 H 26 B25 n-C₉H₁₉CO 0 0 CONH₂ 0 H 27 B26n-C₁₃H₂₇CO 0 0 CO₂Me 0 H 28 B27 n-C₁₃H₂₇CO 0 0 H 1 CO₂ H 29 B28n-C₉H₁₉CO 1 0 H 1 CO₂ H 30 B29 n-C₅H₁₁CO 0 0 CONH₂ 0 H 31 B30 R₁ = R₃ =n-C₉H₁₉CO 0 0 CONH₂ 0 H R₂ = n-C₅H₁₁CO 32 B31 n-C₅H₁₁CO 0 0 H 0 H 33 B32R₁ = n-C₁₃H₂₇CO 0 0 H 0 H R₂ = n-C₁₇H₃₅CO R₃ = n-C₁₅H₃₁CO 34 B34n-C₅H₁₁CO 0 0 CO₂H 0 H 35 B35 R₁ = n-C₅H₁₁CO 0 0 CO₂H 0 H R₂ = R₃ =n-C₉H₁₉CO 36 B36 R₁ = R₃ = n-C₉H₁₉CO 0 0 CO₂H 0 H R₂ = n-C₅H₁₁CO 37 B37R₁ = R₂ = n-C₉H₁₉CO 0 0 CO₂H 0 H R₃ = n-C₅H₁₁CO 38 B38 R₁ = n-C₉H₁₁CO 00 CO₂H 0 H R₂ = R₃ = n-C₅H₁₁CO 39 B39 R₁ = R₃ = n-C₅H₁₁CO 0 0 CO₂H 0 HR₂ = n-C₉H₁₉CO 40 B40 R₁ = R₂ = n-C₅H₁₁CO 0 0 CO₂H 0 H R₃ = n-C₉H₁₉CO 41B41 R₁ = R₃ = n-C₉H₁₉CO 0 1 OH 0 H R₂ = n-C₅H₁₁CO 42 B42 n-C₉H₁₁CO 0 2CO₂H 0 H 43 B43 R₁ = n-C₁₃H₂₇CO 0 0 CO₂H 0 H R₂ = n-C₁₁H₂₃CO R₃ = H

EXAMPLE 1 Preparation of (R)-3-N-Alkanoyloxytetradecanoic Acids (4).

[0124] (1) A solution of methyl 3-oxotetradecanoate (19 g, 0.074 mol) inMeOH (100 mL) was degassed by sparging with argon (15 min).[(R)-Ru(Binap)Cl]₂·NEt₃ catalyst (0.187 g, 0.111 mmol) and 2 N aqueousHCl (0.5 mL) were added and the resulting mixture was hydrogenated at 60psig and 40-50° C. for 18 h. The reaction was diluted with hexanes (250mL), filtered through a short column of silica gel, and concentrated.The crude product was dissolved in tetrahydrofuran (THF; 200 mL),treated 2.4 N aqueous LiOH (83 mL, 0.2 mol) and stirred vigorously atroom temperature for 4 h. The resulting slurry was partitioned betweenether (200 mL) and 1 N aqueous HCl (200 mL) and the layers separated.The aqueous layer was extracted with ether (100 mL) and the combinedethereal extracts were dried (Na₂SO₄) and concentrated. The crudehydroxy acid was dissolved in hot acetonitrile (250 mL), treated withdicyclohexylamine (DCHA; 17 mL, 0.085 mol) and stirred at 60° C. for 1h. The product that crystallized upon cooling was collected andrecrystallized from acetonitrile (650 mL) to yield 28.6 g (91%) ofdicyclohexylammonium (R)-3-hydroxytetradecanoate as a colorless solid:mp 94-95° C.; ¹H NMR (CDCl₃) δ 0.88 (˜t, 3H, J˜6.5 Hz), 1.05-1.58 (m,24H), 1.65 (m, 2H), 1.80 (m, 4H), 2.01 (br d, 4H) 2.18 (dd, 1H, J=15.7,9.4 Hz), 2.36 (dd, 1H, J=15.7, 2.6 Hz), 2.94 (m, 2H), 3.84 (m, 1H)

[0125] (2) To a mixture of the compound prepared in (1) above (50 g,0.117 mol) and 2,4′-dibromoacetophenone (39 g, 0.14 mol) in EtOAc (2.3L) was added triethylamine (19.6 mL, 0.14 mol) and the resultingsolution was stirred for 18 h at room temperature. The voluminousprecipitate that formed was collected and triturated with warm EtOAc(3×400 mL). The combined triturates and filtrate were washed with 1 Maq. HCl, saturated aq. NaCl and dried (Na₂SO₄). Volatiles were removedunder reduced pressure and the crude product obtained was crystallizedfrom EtOAc-hexanes to give 47.2 g (91%) of (R)-3-hydroxytetradecanoicacid p-bromophenacyl ester as a colorless solid: mp 109-109.5° C.; ¹HNMR (CDCl₃) δ 0.88 (˜t, 3H, J˜6.5 Hz) 1.15-1.70 (m, 20H), 2.56 (dd, 1H,J=15.1, 9.1 Hz), 2.69 (dd, 1H, J=15.1, 2.9 Hz), 3.27 (br s, 1H), 4.12(m, 1H), 5.31 (d, 1H, J=16.5 Hz), 5.42 (d, 1H, J=16.5 Hz), 7.65 (d, 2H,J=8.5 Hz), 7.78 (d, 2H, J=8.5 Hz).

[0126] (3) A solution of the compound prepared in (2) above (4.6 g, 10.4mmol) in CH₂Cl₂ (50 mL) containing 4-dimethylaminopyridine (0.12 g, 1.0mmol) and pyridine (5 mL, 62 mmol) was treated at room temperature withmyristoyl chloride (3.1 mL, 11.4 mmol). After stirring for 5 h at roomtemperature MeOH (0.5 mL) was added, and the reaction mixture wasconcentrated. The residue was partitioned between Et₂O (150 mL) and cold10% aqueous HCl (50 mL) and the layers separated. The ethereal layer wasdried (Na₂SO₄) and concentrated and the residue obtained was purified ona short pad of silica gel with 5% EtOAc-hexanes. The diester wasdissolved in AcOH (42 mL) and treated with three equal portions of zincdust (˜6 g, 90 mmol) at 60° C. over a 1 h period. After an additionalhour at 60° C., the cooled reaction mixture was sonicated (5 min),filtered through Celite® and concentrated. The residue was purified byflash chromatography on silica gel with 10% EtOAc-hexanes to give 4.17 g(82%) of (R)-3-tetradecanoyloxytetradecanoic acid as a colorless solid:mp 28-29° C.; ¹H NMR (CDCl₃) δ 0.88 (˜t, 6H), 1.15-1.40 (m, 38H),1.50-1.70 (m, 4H), 2.28 (t, 2H, J=7.4 Hz), 2.56 (dd, 1H, J=15.9, 5.8Hz), 2.63 (dd, 1H, J=15.9, 7.1 Hz), 5.21 (m, 1H).

[0127] (4) In the same manner as described in Example 1-(3), thecompound prepared in Example 1-(2) (2.5 g, 5.68 mmol) was acylated withlauroyl chloride (1.45 mL, 6.25 mmol) in the presence of pyridine (0.57mL, 7.0 mmol) in CH₂Cl₂ (60 mL) and then deprotected with zinc (9.3 g,142 mmol) in AcOH (40 mL) to afford (R)-3-dodecanoyloxytetradecanoicacid as a colorless oil: ¹H NMR (CDCl₃) δ 0.90 (t, 6H, J=6.5 Hz),1.0-1.75 (m, 46H), 2.30 (m, 2H), 2.62 (m, 2H), 5.22 (m, 1H).

[0128] (5) A solution of the compound prepared in Example 1-(2) (2.5 g,5.68 mmol) was treated with undecanoic acid (1.16 g, 6.25 mmol) and EDCMel (2.08 g, 7.0 mmol) in CH₂Cl₂ (60 mL) and then deprotected asdescribed in Example 1-(3) with zinc (9.3 g, 142 mmol) in AcOH (40 mL)to afford (R)-3-undecanoyloxytetradecanoic acid as a colorless oil: ¹HNMR (CDCl₃) δ 0.89 (t, 6H, J=6.7 Hz), 1.0-1.75 (m, 44H), 2.29 (m, 2H),2.61 (m, 2H), 5.22 (m, 1H).

[0129] (6) In the same manner as described in Example 1-(3), thecompound prepared in Example 1-(2) (4.4 g, 10 mmol) was acylated withdecanoyl chloride (2.3 mL, 11 mmol) in the presence of pyridine (1.2 mL,15.0 mmol) in CH₂Cl₂ (100 mL) and then deprotected with zinc (16.4 g,250 mmol) in AcOH (60 mL) to afford (R)-3-decanoyloxytetradecanoic acidas a colorless oil: ¹H NMR (CDCl₃) δ 0.89 (t, 6H, J=6.8 Hz), 1.0-1.75(m, 34H), 2.29 (t, 2H, J=7.4 Hz), 2.61 (t, 2H, J=4.2 Hz), 5.22 (m, 1H).

[0130] (7) In the same manner as described in Example 1-(3), thecompound prepared in Example 1-(2) (2.5 g, 5.68 mmol) was acylated withnonanoyl chloride (1.13 mL, 6.25 mmol) in the presence of pyridine (0.57mL, 7.0 mmol) in CH₂Cl₂ (60 mL) and then deprotected with zinc (9.3 g,142 mmol) in AcOH (40 mL) to afford (R)-3-nonanoyloxytetradecanoic acidas a colorless oil: ¹H NMR (CDCl₃) δ 0.89 (t, 6H, J=6.9 Hz), 1.0-1.75(m, 32H), 2.29 (t, 2H, J=7.5 Hz), 2.61 (m, 2H), 5.22 (m, 1H).

[0131] (8) In the same manner as described in Example 1-(3), thecompound prepared in Example 1-(2) (2.5 g, 5.68 mmol) was acylated withoctanoyl chloride (1.07 mL, 6.25 mmol) in the presence of pyridine (0.57mL, 7.0 mmol) in CH₂Cl₂ (60 mL) and then deprotected with zinc (9.3 g,142 mmol) in AcOH (40 mL) to afford (R)-3-octanoyloxytetradecanoic acidas a colorless oil: ¹H NMR (CDCl₃) δ 0.92 (t, 6H, J=6.9 Hz), 1.0-1.75(m, 30H), 2.32 (t, 2H, J=7.4 Hz), 2.63 (t, 2H, J=4.4 Hz), 5.23 (m, 1H).

[0132] (9) In the same manner as described in Example 1-(3), thecompound prepared in Example 1-(2) (2.5 g, 5.68 mmol) was acylated withheptanoyl chloride (0.97 mL, 6.25 mmol) in the presence of pyridine(0.57 mL, 7.0 mmol) in CH₂Cl₂ (60 mL) and then deprotected with zinc(9.3 g, 142 mmol) in AcOH (40 mL) to afford(R)-3-heptanoyloxytetradecanoic acid as a colorless oil: ¹H NMR (CDCl₃)δ 0.89 (t, 6H, J=6.8 Hz), 1.0-1.75 (m, 28H), 2.29 (t, 2H, J=7.4 Hz),2.61 (d, 2H, J=5.8 Hz), 5.22 (m, 1H).

EXAMPLE 2 (B1) Preparation of3-hydroxy-(S)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O—phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0133] (1) To a solution of 2-(trimethylsilyl)ethyl2-amino-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside (6.46 g, 20.2mmol) in CHCl₃ (300 mL) was added 1 N aqueous NaHCO₃ (300 mL) and2,2,2-trichloroethyl chloroformate (8.5 g, 40 mmol). The resultingmixture was stirred vigorously for 3 h at room temperature. The organiclayer was separated, dried (Na₂SO₄) and concentrated to give a colorlesssyrup. Flash chromatography on silica gel (gradient elution, 30→40%EtOAc-hexanes) afforded 9.6 g (96%) of 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless solid: mp 69-70° C.; ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.94(m, 2H), 1.44 and 1.52 (2s, 6H), 2.94 (br s, 1H), 3.23-3.37 (m, 2H),3.48-3.62 (m, 2H), 3.79 (t, 1H, J=-10.5 Hz), 3.88-4.08 (m, 3H), 4.65 (d,1H, J=8.3 Hz), 4.74 (m, 2H), 5.39 (d, 1H, J=7.4 Hz).

[0134] (2) A solution of the compound prepared in (1) above (7.5 g, 15.2mmol), (R)-3-tetradecanoyloxytetradecanoic acid (7.58 g, 16.7 mmol) and4-pyrrolidinopyridine (0.25 g, 1.7 mmol) in CH₂Cl₂ (95 mL) was treatedwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC-MeI;4.94 g, 16.7 mmol) and stirred for 16 h at room temperature. Thereaction mixture was filtered through a short pad of Celite®,concentrated, and the resulting residue was heated at 60° C. in 90%aqueous AcOH (100 mL) for 1 h. The mixture was concentrated and residualAcOH and water were removed by azeotroping with toluene (2×150 mL). Thecrude diol was purified by flash chromatography on silica gel (gradientelution, 30→40% EtOAc-hexanes) to give 11.8 g (83%) of2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.9 (m, 8H),1.1-1.7 (m, 42H), 2.30 (t, 2H, J=7.4 Hz), 2.52 (m, 2H), 3.36-3.72 (m,4H), 3.78-4.03 (m, 3H), 4.57 (d, 1H, J=8.3 Hz), 4.65 (d, 1H, J=1 Hz),4.77 (d, 1H, J=1 Hz), 5.0-5.15 (m, 2H), 5.20 (d, 1H, J=7.4 Hz).

[0135] (3) A solution of the compound prepared in (2) above (10.9 g, 12mmol) and pyridine (2 mL, 25 mmol) in CH₂Cl₂ (125 mL) at 0° C. wastreated dropwise over 15 min with a solution of2,2,2-trichloro-1,1-dimethylethyl chloroformate (3.17 g, 13.2 mmol) inCH₂Cl₂ (25 mL). The reaction mixture was allowed to warm slowly toambient temperature over 3.5 h. 4-Pyrrolidinopyridine (0.89 g, 6.0mmol), N,N-diisopropylethylamine (10.5 mL, 60 mmol) and diphenylchlorophosphate (3.7 mL, 18 mmol) were added sequentially and theresulting mixture was stirred for 5 h at room temperature. The reactionmixture was diluted with CH₂Cl₂ (500 mL), washed with cold 7.5% aqueousHCl (2×250 mL), water (250 mL), saturated aqueous NaHCO₃ (250 mL), dried(Na₂SO₄), and then concentrated. The residue obtained was purified byflash chromatography on silica gel eluting with 12.5% EtOAc-hexanes togive 15.1 g (95%) of 2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichlorethoxycarbonylamino)-β-D-glucopyranosideas a viscous oil: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.8-1.0 (m, 8H),1.1-1.65 (m, 42H), 1.83 and 1.90 (2s, 6H), 2.15-2.45 (m, 4H), 3.34 (q,1H, J=8 Hz), 3.37 (m, 1H), 3.81 (m, 1H), 3.95 (m, 1H), 4.27 (dd, 1H,J=12, 5 Hz), 4.34 (d, 1H, J=12 Hz), 4.58 (d, 1H, J=12 Hz), 4.66 (q, 1H,J=9 Hz), 4.86 (d, 1H, J=12 Hz), 5.03 (d, 1H, J=7.9 Hz), 5.21 (m, 1H),5.54-5.70 (m, 2H), 7.2-7.8 (m, 10H).

[0136] (4) A solution of the compound prepared in (3) above (1.87 g,1.41 mmol) in CH₂Cl₂ (3 mL) at 0° C. was treated dropwise over 10 minwith trifluoroacetic acid (TFA; 6 mL) and then stirred for 4 h at 0° C.The reaction mixture was concentrated and residual TFA was removed byazeotroping with toluene (2×5 mL). A solution of the lactol anddimethylformamide (2.2 mL, 28.2 mmol) in CH₂Cl₂ (14 mL) at 0° C. wastreated with oxalyl bromide (2.0 M in CH₂Cl₂; 2.1 mL, 4.2 mmol) dropwiseover 15 min and the resulting suspension was stirred at 0° C. for 24 h.The reaction mixture was partitioned between cold saturated aqueousNaHCO₃ (25 mL) and ether (50 mL) and the layers were separated. Theethereal layer was washed with saturated aqueous NaCl, dried (Na₂SO₄)and concentrated to give 1.85 g (˜100%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylbromide as a colorless glass.

[0137] (5) A solution of (R)-2-amino-3-benzyloxy-1-propanol (0.46 g,2.33 mmol) and (R)-3-tetradecanoyloxytetradecanoic acid (1.29 g, 2.83mmol) in CH₂Cl₂ (20 mL) was treated with EDC·MeI (0.78 g, 2.79 mmol) andstirred for 16 h at room temperature. The reaction mixture was filteredthrough a short pad of Celite® and concentrated. Flash chromatography onsilica gel with 45% EtOAc-hexanes afforded 1.1 g (69%) of3-benzyloxy-(R)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propanol asa colorless solid: mp 42-44.5° C.;

[0138]¹H NMR δ 0.88 (t, 6H, J=6.5 Hz), 1.0-1.7 (m, 42H), 2.50 (t, 2H,J=7.5 Hz), 2.46 (m, 2H), 3.56 (br s, 1H), 3.5-3.75 (m, 3H), 3.78 (dd,1H, J=11,4 Hz), 4.08 (m, 1H), 4.51 (s, 2H), 5.17 (m, 1H), 6.36 (d, 1H,J=7.8 Hz), 7.2-7.4 (m, 5H).

[0139] (6) To a solution of the compound prepared in (4) above (1.00 g,0.776 mmol) and the compound prepared in (5) above (0.35 g, 0.57 mmol)in dichloroethane (4.5 mL) was added powdered 4 A molecular sieves (1.25g) and calcium sulfate (2.7 g, 20 mmol). After stirring for 10 min atroom temperature, the mixture was treated with mercury cyanide (1.0 g,4.0 mmol) and then heated to reflux for 12 h shielded from light. Thereaction mixture was diluted with CH₂Cl₂ (25 mL) and filtered through apad of Celite®. The filtrate was washed with 1 N aqueous KI (25 mL),dried (Na₂SO₄) and concentrated. The residue was chromatographed onsilica gel with EtOAc-hexanes-MeOH (80:20:0→70:30:1, gradient elution)to give 0.66 g (63%) of3-benzyloxy-(S)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-3-O-[(R)-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR δ 0.88 (t, 12H, J=˜6.5 Hz), 1.0-1.65 (m,84H), 1.79 and 1.86 (2s, 6H), 2.1-2.5 (m, 8H), 3.35-3.55 (m, 3H),3.65-3.8 (m, 3H), 4.1-4.75 (m, 9H), 5.05-5.3 (m, 2H), 5.3-5.5 (m, 2H),6.04 (d, 1H, J=8.4 Hz), 7.05-7.45 (m, 15H).

[0140] (7) A stirred solution of the compound prepared in (6) above(0.60 g, 0.328 mmol) in AcOH (9 mL) at 55° C. was treated with zinc dust(1.1 g, 16 mmol) in three equal portions over 1 h. The cooled reactionmixture was sonicated, filtered through a bed of Celite® andconcentrated. The resulting residue was partitioned between CH₂Cl₂ (60mL) and cold 1 N aqueous HCl (35 mL) and the layers separated. Theorganic layer was washed with 5% aqueous NaHCO₃, dried (Na₂SO₄) andconcentrated. A mixture of the residue obtained and(R)-3-tetradecanoyloxytetradecanoic acid (0.18 g, 0.39 mmol) in CH₂Cl₂(3.5 mL) was stirred with powdered 4A molecular sieves (0.1 g) for 30min at room temperature and then treated with2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ; 0.12 g, 0.49mmol). The resulting mixture was stirred for 6 h at room temperature,filtered through Celite® and then concentrated. Chromatography on silicagel (gradient elution, 0.5→1% MeOH—CHCl₃) afforded 0.31 g (50%) of3-benzyloxy-(S)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 18H, J=˜6.5 Hz),1.0-1.8 (m, 126H),2.1-2.5 (m, 12H), 3.35-3.75 (m, 6H),3.80 (m, 2H),4.23(m, 1H), 4.46 (d, 1H, J=12 Hz), 4.51 (d, 1H, J=12 Hz), 4.65 (q, 1H,J=˜9.5 Hz), 4.82 (d, 1H, J=8.1 Hz),5.05-5.25 (m, 3H), 5.47 (t, 1H,J=˜9.5 Hz), 6.16 (d, 1H, J=8.1 Hz), 6.31 (d, 1H, J=8.4 Hz),7.1-7.4 (m,15H).

[0141] (8) A solution of the compound prepared in (7) above (0.26 g,0.138 mmol) in THF (25 mL) was hydrogenated in the presence of 5%palladium on carbon (50 mg) at room temperature and atmospheric pressurefor 16 h. After removal of the catalyst by filtration, AcOH (3 mL) andplatinum oxide (0.14 g) were added and the hydrogenation was continuedat room temperature and 75 psig for 24 h. The resulting opalescentreaction mixture was diluted with 2:1 CHCl₃—MeOH (20 mL) and sonicatedbriefly to give a clear solution. The catalyst was collected, washedwith 2:1 CHCl₃—MeOH (2×5 mL) and the combined filtrate and washings wereconcentrated. The residue was dissolved in 1% aqueous triethylamine (10mL) by sonicating for 5 min at 35° C. and the resulting solution waslyophilized. Flash chromatography on silica gel withchloroform-methanol-water-triethylamine (94:6:0.5:0.5→88:12:1.0:1.0,gradient elution) afforded 0.20 g (84%) of product as a colorlesspowder. A portion of the chromatography product (0.166 g) was dissolvedin cold 2:1 CHCl₃—MeOH (33 mL) and washed with cold 0.1 N aqueous HCl(14 mL). The lower organic layer was filtered and concentrated and thefree acid obtained was lyophilized from 1% aqueous triethylamine(pyrogen free, 15 mL) to give 0.160 g of3-hydroxy-(S)-2-[(R)-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a colorless solid: mp 178-180° C. (dec); IR(film) 3293, 3103, 2959, 2924, 2855, 1732, 1654, 1640, 1553, 1467, 1377,1259, 1175, 1106, 1086, 1050, 803, 720 cm⁻¹; HMR(CDCl₃—CD₃OD) δ 0.88 (t,18H, J=˜7 Hz), 1.0-1.7 (m, 135H), 2.15-2.75 (m, 12H), 3.02 (q, 6H, J=7Hz), 3.35-4.1 (m, 7H), 4.22 (q, 1H, J=˜9.5 Hz), 4.77 (d, 1H, J=8 Hz),5.05-5.35 (m, 4H), 6.58 (d, 1H, J=6 Hz),6.73 (d, 1H, J=7.5 Hz, NH); ¹³CNMR (CDCl₃) δ 173.5, 173.2, 170.7, 170.5, 170.0, 100.7, 75.9, 72.7,71.2, 71.0, 70.8, 70.6, 67.9, 61.7, 60.5, 55.0, 50.4, 45.6, 41.4, 39.5,34.5, 34.4, 32.0, 31.8, 30.3, 29.8, 29.4, 29.3, 25.3, 25.1, 22.7, 14.2,8.6.

[0142] Anal. Calcd for C₁₉H₁₉₂N_(3 O) ₁₈P·5H₂O: C, 64.84; H, 11.10; N,2.29; P, 1.69. Found: C, 64.69; H, 11.24; N, 1.93; P, 1.44.

EXAMPLE 3 (B2) Preparation of3-hydroxy-(R)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O—phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0143] (1) A solution of the compound prepared in Example 2-(5) (0.63 g,1.02 mmol) in CH₂Cl₂ (7 mL) was treated sequentially with pyridine (0.4mL, 5 mmol), 4-dimethylaminopyridine (cat.) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (0.307 g, 1.23 mmol) andstirred for 16 h at room temperature. The reaction mixture was dilutedwith CH₂Cl₂ (25 mL), washed with saturated aqueous NaHCO₃ (25 mL) anddried (Na₂SO₄). Removal of volatiles in vacuo gave a residue that wasdissolved in THF-AcOH (10 mL, 9:1) and hydrogenated in the presence of5% palladium on carbon (150 mg) at room temperature and atmosphericpressure for 24 h. After removal of the catalyst by filtration andconcentration of the filtrate, the residue was purified by flashchromatography on silica gel with 35% EtOAc-hexanes to give 0.536 g(72%) of3-(2,2,2-trichloro-1,1-dimethylethoxycarbonyloxy)-(S)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propanolas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.5 Hz), 1.1-1.7(m, 42H), 1.94 (s, 6H), 2.30 (t, 2H, J=7.5 Hz), 2.47 (d, 2H, J=6 Hz),3.50 (br s, 1H), 3.72 (m, 2H), 4.15-4.35 (m, 3H), 5.15 (m, 1H), 6.18 (d,1H, J=7.2 Hz).

[0144] (2) In the same manner as described in Example 2-(6), thecompound prepared in (1) above (0.310 g, 0.426 mmol) and the compoundprepared in Example 2-(4) (0.961 g, 0.745 mmol) were coupled in thepresence of mercury cyanide (0.43 g, 1.7 mmol) to give 0.644 g (78%) of3-(2,2,2-trichloro-1,1-dimethylethyloxycarbonyloxy)-(S)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-3-O-[(R)-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=˜6.5 Hz),1.0-1.7 (m, 84H), 1.81 and 1.89 (2s, 6H), 1.93 (s, 6H), 2.15-2.55 (m,8H), 3.45-3.7 (m, 2H), 3.80 (br d, 1H, J=9 Hz), 3.9-4.45 (m, 6H),4.6-4.8 (m, 3H), 4.87 (d, 1H, J=8.1 Hz), 5.0-5.25 (m, 2H), 5.48 (t, 1H,J=˜9.5 Hz), 6.1-6.3 (m, 2H).

[0145] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (0.602 g, 0.310 mmol) was deprotectedwith zinc (1.5 g, 23 mmol) and acylated with(R)-3-tetradecanoyloxytetradecanoic acid, (0.17 g, 0.37 mmol) in thepresence of EEDQ (0.115 g, 0.467 mmol) to give 0.365 g (66%) of3-hydroxy-(R)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 18H, J=6.5 Hz), 1.0-1.7(m, 126H), 2.15-2.55 (m, 12H), 3.18 (br s, 1H), 3.45-3.8 (m, 8H),3.85-4.05 (m, 2H), 4.69 (q, 1H, J=9.5 Hz), 5.05-5.25 (m, 3H), 5.42 (t,1H, J=9.5 Hz), 6.42 (d, 1H, J=7.8 Hz), 6.59 (d, 1H, J=7.2 Hz), 7.1-7.4(m, 10H).

[0146] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (0.355 g, 0.196 mmol) was hydrogenated inthe presence of platinum oxide (175 mg) to give 0.265 g (77%) of3-hydroxy-(R)-2-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a colorless solid: mp 159-160° C.; IR (film)3291, 2956, 2922, 2853, 1738, 1732, 1716, 1650, 1643, 1556, 1468, 1171,1109, 1083, 1051 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=˜6.5 Hz),1.0-1.7 (m, 135H), 2.15-2.75 (m, 12H), 3.06 (q, 6H, J=7 Hz), 3.25-3.45(m, 2H), 3.5-4.05 (m, 12H), 4.19 (q, 1H, J=˜9.5 Hz), 4.48 (d, 1H, J=8.4Hz), 5.04-5.26 (m, 4H), 7.18 (d, 1H, J=7.8 Hz), 7.27 (d, 1H, J=8.7 Hz);¹³C NMR (CDCl₃) δ 173.5, 173.4, 170.7, 170.6, 170.1, 101.0, 76.0, 72.6,71.4, 71.0, 70.8, 70.6, 68.7, 61.8, 60.5, 55.3, 50.5, 45.6, 41.5, 41.4,39.5, 34.6, 34.4, 34.3, 32.0, 29.8, 29.4, 25.4, 25.1, 22.7, 14.1, 8.6.

[0147] Anal. Calcd for C₉₉H₁₉₂N₃O₁₈P·H₂O: C, 67.50; H, 11.10; N, 2.39;P, 1.76. Found: C, 67.40; H, 11.22; N, 2.34; P, 2.11.

EXAMPLE 4 (B3) Preparation of3-hydroxy-(S)-2-[(R)-3-dodecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₁H₂₃CO, X=Y=O,N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0148] (1) A solution of D-glucosamine hydrochloride (20 g, 92.8 mmol)in H₂O (250 mL) was treated with a saturated aqueous NaHCO₃ (250 mL) and2,2,2-trichloroethyl chloroformate (14.05 mL, 102 mmol) and stirredvigorously for 18 h. The white solid that formed was collected on afritted funnel and dried under vacuum for 24 h. A solution of the solidin pyridine (100 mL) was cooled to 0° C. and treated with aceticanhydride (100 mL) via addition funnel. The solution was stirred for 18h at room temperature, poured into 1 L of H₂O and extracted with CHCl₃(3×500 mL). The solvent was removed in vacuo to afford 45 g (quant.) ofN-(2,2,2-trichloroethoxycarbonylamino)-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranosidewhich was used without further purification: ¹H NMR (CDCl₃) δ 2.06 (s,6H), 2.12 (s, 3H), 2.22 (s, 3H), 4.03 (m, 1H), 4.07 (d, 1H, J=12.4 Hz),4.22 (dt, 1H, J=9.9, 3.6 Hz), 4.30 (dd, 1H, J=12.4, 4.0 Hz), 4.64 (d,1H, J=9.6 Hz), 5.28 (dt, 1H, J=10.2, 9.9 Hz), 6.25 (d, 1H, J=3.6 Hz).

[0149] (2) A solution of (R)-2-amino-3-benzyloxy-1-propanol (5 g, 27.6mmol) in CH₂Cl₂ (250 mL) was treated with allyl chloroformate (3.2 mL,30 mmol) and saturated aqueous NaHCO₃ (250 mL) for 18 h. The organiclayer was separated and concentrated in vacuo. Purification bychromatography eluting with 30% EtOAc/hexanes afforded 6.9 g (94%) of(R)-2-(allyloxycarbonylamino)-3-benzyloxy-1-propanol as an amorphoussolid: ¹H NMR (CDCl₃) δ 2.56 (br s, 1H), 3.69 (m, 3H), 388 (m, 2H), 4.54(s, 2H), 4.58 (d, 2H, J=5.6 Hz), 5.23 (dd, 1H, J=10.4, 1.1 Hz), 5.33(dd, 1H, J=17.1, 1.1 Hz), 5.42 (m, 1H), 5.93 (m, 1H), 7.35 (m, 5H).

[0150] (3) A solution of the compounds prepared in (1) and (2) above(8.9 g, 17 mmol and 3.6 g, 10 mmol, respectively) in CH₂Cl₂ was treatedwith boron trifluoride etherate (4.3 mL, 34 mmol) at room temperaturefor 16 h. The reaction mixture was quenched with saturated aq. NaHCO₃(100 mL) and extracted with EtOAc (3×100 mL). The combined EtOAcextracts were dried (Na₂SO₄) and concentrated. The residue obtained waschromatographed with 20% EtOAc/hexanes to afford 6.03 g (83%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-3,4,6-tri-O-acetyl-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 2.02 (s, 3H), 2.03 (s, 3H), 2.08(s, 3H), 3.45 (m, 1H), 3.54 (m, 1H), 3.64 (m, 1H), 3.76 (d, 1H, J=7.2Hz), 3.91 (m, 2H), 4.12 (d, 1H, J=12.2 Hz), 4.26 (dd, 1H, J=12.4, 4.7Hz), 4.37 (d, 1H, J=8.2 Hz), 4.43 (d, 1H, J=12.1 Hz), 4.55 (m, 2H), 4.68(m, 2H), 4.87 (d, 1H, J=8.0 Hz), 5.07 (m, 2H), 5.21 (d, 1H, J=9.7 Hz),5.29 (d, 1H, J=17.3 Hz), 5.91 (m, 1H), 7.36 (m, 5H).

[0151] (4) A solution of the compound prepared in (3) above (6.0 g, 8.3mmol) in methanol (83 mL) was treated with ammonium hydroxide (8.3 mL)at room temperature for 2 h. The solvent was removed in vacuo andreplaced with 2,2-dimethoxypropane (50 mL) and camphorsulfonic acid (100mg) was added. The reaction was stirred for 18 h, neutralized with solidNaHCO₃ (1 g), filtered and concentrated in vacuo. Purification bychromatography with 50% EtOAc/hexanes afforded 4.58 g (86%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside:¹H NMR (CDCl₃) δ 1.46 (s, 3H), 1.53 (s, 3H), 2.94 (m, 1H), 3.25 (m, 1H),3.55 (m, 4H), 3.83 (m, 3H), 3.93 (m, 3H), 4.52 (m, 5H), 4.68 (d, 1H,J=12.1 Hz), 4.77 (d, 1H, J=12.1 Hz), 5.07 (m, 1H), 5.26 (m, 2H), 5.92(m, 1H), 7.37 (m, 5H).

[0152] (5) A solution of the compound prepared in (4) above (1.0 g, 1.56mmol) in CH₂Cl₂ (20 mL) was treated with(R)-3-dodecanoyloxytetradecanoic acid (730 mg, 1.71 mmol) in thepresence of EDC·MeI (560 mg, 1.87 mmol) and 4-pyrrolidinopyridine (50mg). The reaction was stirred at room temperature for 18 h and filteredthrough a 6×8 cm plug of silica gel using 20% EtOAc/hexanes as eluent toafford 1.33 g (82%) of 3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.8 Hz), 1.1-1.6(m, 38H), 1.37 (s, 3H), 1.46 (s, 3H), 2.28 (t, 2H, J=7.4 Hz), 2.49 (dd,1H, J=15.1, 6.0 Hz), 2.61 (dd, 1H, J=15.1, 6.6 Hz), 3.25-4.0 (m, 9H),4.38 (m, 2H), 4.54 (m, 2H), 4.65 (m, 2H), 4.97 (m, 2H), 5.25 (m, 5H),5.88 (m, 1H), 7.34 (m, 5H).

[0153] (6) To a solution of the compound prepared in (5) above (1.31 g,1.25 mmol) in THF (20 mL) was added dimethyl malonate (1.0 mL, 8.8 mmol)and the solution was degassed in a stream of argon for 30 min.Tetrakis(triphenylphosphine)palladium(0) (200 mg) was added and thereaction was stirred at room temperature for 2 h, and then concentratedin vacuo. The residue obtained was chromatographed on silica gel elutingwith 5-10% EtOAc/CHCl₃. The free amine obtained was acylated with(R)-3-dodecanoyloxytetradecanoic acid (560 mg, 1.38 mmol) in thepresence of EEDQ (370 mg, 1.5 mmol) in CH₂Cl₂ (15 mL). After stirring atroom temperature for 18 h, the solvent was removed in vacuo and theresultant oil was chromatographed on silica gel eluting with 20%EtOAc/hexanes to afford 1.02 g (63%) of3-benzyloxy-(S)-2-[(R)-3-dodecanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.9Hz), 1.1-1.7 (m, 78H), 1.38 (s, 3H), 1.46 (s, 3H), 2.26 (m, 4H), 2.49(dd, 1H, J=15.1, 6.0 Hz), 2.61 (dd, 1H, J=15.1, 6.6 Hz), 3.25-4.0 (m,9H), 5.01 (m, 2H), 6.02 (d, 1H, J=8.4 Hz), 7.34 (m, 5H).

[0154] (7) The compound prepared in (6) above (1.0 g, 0.78 mmol) wastreated with 90% aqueous AcOH (20 mL) for 1 h at 60° C. The solution wasconcentrated in vacuo and residual AcOH and H₂O were removed byazeotroping with toluene (10 mL). The residue was dissolved in CH₂Cl₂,cooled to 0° C., and treated with pyridine (0.076 mL, 0.94 mmol) and asolution of 2,2,2-trichloro-1,1-dimethylethyl chloroformate (205 mg,0.86 mmol) in CH₂Cl₂ (5 mL). The reaction mixture was then allowed towarm and stir at room temperature for 18 h. The resulting light yellowsolution was treated with diphenyl chlorophosphate (0.24 mL, 1.17 mmol),triethylamine (0.22 mL, 1.56 mmol) and catalytic 4-pyrrolidinopyridine(50 mg), and then stirred an additional 24 h at room temperature. Thereaction mixture was diluted with Et₂O (100 mL) and washed with 10% aq.HCl (50 mL). The organic phase was separated, dried over Na₂SO₄ andconcentrated in vacuo. Chromatography over silica gel using 10%EtOAc/hexanes afforded 1.13 g (85%) of3-benzyloxy-(S)-2-[(R)-3-dodecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.9Hz), 1.1-1.6 (m, 78H), 1.78 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H),2.18(m,3H),2.40(m, 2H),2.67(m, 1H), 2.88 (d, 1H, J=6.6 Hz), 2.97 (d, 1H, J=6.9Hz), 3.41 (m, 2H), 3.72 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H), 4.42 (d,1H, J=11.8 Hz), 4.64 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75 (d, 1H,J=4.3 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.23 (m, 15H).

[0155] (8) In the same manner as described in Example 2-(7), thecompound prepared in (7) above (1.1 g, 0.65 mmol) was deprotected withzinc (2.1 g, 32 mmol) and acylated with (R)-3-dodecanoyloxytetradecanoicacid (330 mg, 0.78 mmol) in the presence of EEDQ (230 mg, 0.94 mmol) toafford 399 mg (37%) of3-benzyloxy-(S)-2-[(R)-3-dodecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0156] (9) In the same manner as described in Example 2-(8), thecompound prepared in (8) above (399 mg, 0.24 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 65 mg (16%) of3-hydroxy-(S)-2-[(R)-3-dodecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 181-184° C. (dec): IR (film)3306, 2956, 2922, 2852, 1732, 1644, 1549, 1467, 1377, 1164, 1106, 1051,721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.7 Hz), 1.1-1.7 (m,123H), 2.2-2.7 (m, 12H), 3.06 (q, 6H, J=7.1 Hz), 3.3-4.0 (m, 13H), 4.23(m, 1H), 4.44 (d, 1H, J=7.7 Hz), 5.0-5.3 (m, 4H); ¹³C NMR (CDCl₃) δ173.9, 173.5, 173.3, 170.8, 170.5, 170.1, 101.0, 75.5, 73.0, 71.1, 70.9,70.6, 67.9, 61.6, 60.7, 54.4, 50.4, 45.8, 41.6, 41.4, 39.6, 34.6, 31.9,29.7, 29.4, 29.3, 25.4, 25.1, 22.7, 14.2, 8.6.

[0157] Anal. Calcd. for C₉₃H₁₈₀N₃O₁₈P H₂O: C, 66.59; H, 10.94; N, 2.50;P, 1.85. Found: C, 66.79; H, 10.65; N, 2.36; P, 1.70.

EXAMPLE 5 (B4) Preparation of3-hydroxy-(S)-2-[(R)-3-undecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₀H₂₁CO, X═Y=O,N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0158] (1) In the same manner as described in Example 4-(5), thecompound prepared in Example 4-(4) (1.0 g, 1.56 mmol) was acylated with(R)-3-undecanoyloxytetradecanoic acid (705 mg, 1.71 mmol) in thepresence of EDC·MeI (560 mg, 1.87 mmol) and 4-pyrrolidinopyridine (50mg) in CH₂Cl₂ (20 mL) to afford 1.23 g (77%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-undecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ0.88 (t, 6H, =6.9 Hz), 1.1-1.6(m, 36H), 1.37 (s, 3H), 1.46 (s, 3H), 2.28 (m, 2H), 2.52 (dd, 1H,J=15.1, 6.0 Hz), 2.61 (dd, 1H, =15.5, 6.8 Hz), 3.25 (m, 1H), 3.35-4.0(m, 9H), 4.31 (m, 2H), 4.54 (m, 2H), 4.64 (m, 2H), 5.02 (m, 2H), 5.18(m, 2H), 5.25 (m, 1H), 5.86 (m, 1H), 7.34 (m, 5H).

[0159] (2) In the same manner as described in Example 4-(6) the compoundprepared in (1) above (1.21 g, 1.17 mmol) was deprotected in THF (20 mL)in the presence of dimethyl malonate (1.0 mL, 0.88 mmol) andtetrakis(triphenylphosphine)palladium(0) (200 mg) and then acylated with(R)-3-undecanoyloxytetradecanoic acid (540 mg, 1.30 mmol) in thepresence of EEDQ (370 mg, 1.5 mmol) to afford 921 mg (61%) of3-benzyloxy-(S)-2-[(R)-3-undecanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-undecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6Hz), 1.1-1.7 (m, 72H), 1.38 (s, 3H), 1.46 (s, 3H), 2.26 (m, 3H), 2.38(m, 5H), 2.49 (dd, 1H, J=15.2, 6.0 Hz), 2.61 (dd, 1H, J=15.0, 6.5 Hz),3.25-4.0 (m, 9H), 4.30 (m, 2H), 4.59 (m, 3H), 6.03 (d, 1H, J=8.2 Hz),7.34 (m, 5H).

[0160] (3) In the same manner as described in Example 4-(7) the compoundprepared in (2) above (910 g, 0.71 mmol) was deprotected in 90% aqueousAcOH (20 mL), and then treated with pyridine (0.071 mL, 0.88 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (195 mg, 0.80 mmol) inCH₂Cl₂ followed by diphenyl chlorophosphate (0.23 mL, 1.10 mmol),triethylamine (0.20 mL, 1.46 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 1.10 g (89%) of3-benzyloxy-(S)-2-[(R)-3-undecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-undecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.7Hz), 1.1-1.6 (m, 72H), 1.78 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H), 2.18(m, 3H), 2.40 (m, 2H), 2.67 (m, 1H), 2.88 (d, 1H, J=6.7 Hz), 2.97 (d,1H, J=6.9 Hz), 3.41 (m, 2H), 3.72 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H),4.42 (d, 1H, J=11.8 Hz), 4.64 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75(d, 1H, J=4.6 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.22 (m, 15H).

[0161] (4) In the same manner as described in Example 2-(7), thecompound prepared in (3) above (1.0 g, 0.59 mmol) was deprotected withzinc (2.0 g, 30 mmol) and acylated with (R)-3-undecanoyloxytetradecanoicacid (292 mg, 0.71 mmol) in the presence of EEDQ (210 mg, 0.85 mmol) toafford 388 mg (40%) of3-benzyloxy-(S)-2-[(R)-3-undecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0162] (5) In the same manner as described in Example 2-(8), thecompound prepared in (4) above (388 mg, 0.24 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 65 mg (17%) of3-hydroxy-(S)-2-[(R)-3-undecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 183-184° C.; IR (film) 3306,2956, 2922, 2852, 1732, 1644, 1550, 1467, 1377, 1164, 1106, 1052, 721cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.8 Hz), 1.1-1.7 (m, 117H),2.2-2.7 (m, 12H), 3.07 (q, 6H, J=7.1 Hz), 3.3-3.9 (m, 13H), 4.23 (m,1H), 4.45 (d, 1H, J=8.2 Hz), 5.0-5.3 (m, 4H); ¹³C NMR (CDCl₃) δ 173.8,173.5, 173.3, 170.8, 170.5, 170.1, 101.0, 75.5, 73.1, 71.5, 71.3,70.9,70.6, 67.8, 61.6, 60.7, 54.4, 50.5, 45.8, 41.5, 41.4, 39.5, 34.6,34.4, 32.0, 31.2, 29.8, 29.7, 29.4, 28.6, 26.1, 25.4, 25.1, 22.7, 14.1,8.6.

[0163] Anal. Calcd. for C₉₀H₁₇₄N₃O₁₈P H₂O: C, 66.10; H, 10.85; N, 2.57;P, 1.89. Found: C, 66.34; H, 10.69; N, 2.32; P, 1.99.

EXAMPLE 6 (B5) Preparation of3-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O, N=M=Q=0,R₄=R₅=R₇=R₉=H, R₆=OH, P=1 R₈=PO₃H₂).

[0164] (1) In the same manner as described in Example 4-(5), thecompound prepared in Example 4-(4) (2.0 g, 3.12 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (1.36 g, 3.42 mmol) in the presenceof EDC·MeI (1.12 g, 3.74 mmol) and 4-pyrrolidinopyridine (100 mg) inCH₂Cl₂ (40 mL) to afford 2.49 g (79%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.7 Hz), 1.1-1.6(m, 34H), 1.36 (s, 3H), 1.46 (s, 3H), 2.27 (t, 2H, J=6.9 Hz), 2.48 (dd,1H, J=15.1, 6.0 Hz), 2.60 (dd, 1H, J=15.1, 6.7 Hz), 3.25 (m, 1H),3.35-4.0 (m, 9H), 4.23 (m, 1H), 4.42 (m, 1H), 4.52 (m, 4H), 4.95 (m,2H), 5.17 (m, 3H), 5.88 (m, 1H), 7.36 (m, 5H).

[0165] (2) In the same manner as described in Example 4-(6) the compoundprepared in (1) above (2.47 g, 2.42 mmol) was deprotected in THF (40 mL)in the presence of dimethyl malonate (2.0 mL, 1.75 mmol) andtetrakis(triphenylphosphine)palladium(0) (400 mg) and then acylated with(R)-3-decanoyloxytetradecanoic acid (1.06 g, 2.66 mmol) in the presenceof EEDQ (740 mg, 3 mmol) to afford 1.86 g (60%) of3-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR(CDCl₃) δ 0.87 (t, 12H, J=6.7 Hz),1.1-1.7(m, 68H), 1.37 (s, 3H), 1.46 (s, 3H), 2.32 (m, 4H), 2.50 (dd, 1H,J=15.1, 6.0 Hz), 2.62 (dd, 1H, J 15.1, 6.8 Hz), 3.29 (m, 2H), 3.44 (m,1H), 3.55 (m, 1H), 3.74 (m, 3H), 3.93 (m, 1H), 4.18 (m, 1H), 4.34 (m,1H), 4.57 (d, 1H, J=11.8 Hz), 4.65 (m, 2H), 5.01 (m, 2H), 6.04 (d, 1H,J=8.3 Hz), 7.36 (m, 5H).

[0166] (3) In the same manner as described in Example 4-(7) the compoundprepared in (2) above (900 mg, 0.72 mmol) was deprotected in 90% aqueousAcOH (40 mL), and then treated with pyridine (0.071 mL, 0.88 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (195 mg, 0.80 mmol) inCH₂Cl₂ followed by diphenyl chlorophosphate (0.23 mL, 1.10 mmol),triethylamine (0.20 mL, 1.46 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 1.05 g (86%) of3-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.3Hz), 1.1-1.6 (m, 68H), 1.78 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H), 2.18(m, 3H), 2.40 (m, 2H),2.67 (m, 1H), 2.88 (d, 1H, J=6.5 Hz), 2.97 (d, 1H,J=6.9 Hz), 3.41 (m, 2H), 3.72 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H), 4.42(d, 1H, J=11.8 Hz), 4.64 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75 (d,1H, J=4.3 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.22 (m, 15H).

[0167] (4) In the same manner as described in Example 2-(7), thecompound prepared in (3) above (1.0 g, 0.60 mmol) was deprotected withzinc (2.0 g, 30 mmol) and acylated with (R)-3-decanoyloxytetradecanoicacid (285 mg, 0.72 mmol) in the presence of EEDQ (210 mg, 0.86 mmol) toafford 332 mg (34%) of3-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0168] (5) In the same manner as described in Example 2-(8), thecompound prepared in (4) above (332 mg, 0.20 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 173 mg (55%)of 3-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 179-181° C.; IR (film) 3295,2956, 2923, 2853, 1732, 1650, 1555, 1467, 1377, 1320, 1169, 1134, 1104,1051, 979, 801, 721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.9Hz), 1.1-1.7 (m, 111H), 2.2-2.7 (m, 12H), 3.07 (q, 6H, J=6.5 Hz),3.3-4.3 (m, 14H), 4.45 (d, 1H, J=8.0 Hz), 5.0-5.3 (m, 4H), 7.39 (m, 1H),7.53 (d, 1H, J=9.1 Hz); ¹³C NMR (CDCl₃) δ 173.7, 173.4, 173.2, 170.7,170.5, 170.1, 101.0, 75.4, 73.1, 71.6, 71.1, 70.8, 70.5, 67.8, 61.4,60.8, 54.3, 50.4, 45.8, 41.3, 39.5, 34.5, 31.9, 29.8, 29.7, 29.4, 25.4,25.1, 22.7, 14.1, 8.6.

[0169] Anal. Calcd. for C₈₇H₁₆₈N₃O₁₈P H₂O: C, 65.58; H, 10.75; N, 2.64;P, 1.94. Found: C, 65.49; H, 10.75; N, 2.64; P, 1.97.

EXAMPLE 7 (B6) Preparation of3-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-6-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound of R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O, N=M=Q=0,R₄=R₅=R₇=R₈=H, R₆=OH, P=1, R₉=PO₃H₂).

[0170] (1) In the same manner as described in Example 4-(7) the compoundprepared in Example 6-(2) (900 mg, 0.72 mmol) was deprotected in 90%aqueous AcOH (20 mL). The residue was dissolved in CH₂Cl₂ (20 mL),cooled to 0° C., and treated with triethylamine (0.14 mL, 1.0 mmol) anddiphenyl chlorophosphate (0.17 mL, 0.8 mmol). The mixture was stirredfor an additional 6 h, and then quenched with 50 mL of 10% HCl. Theproduct was extracted with EtOAc (3×50 mL) and dried over Na₂SO₄.Chromatography on silica gel with 50% EtOAc/hexanes afforded 636 mg(63%) of 3-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-6-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.0Hz), 1.1-1.6 (m, 68H), 1.79 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H), 2.18(m, 3H), 2.40 (m, 2H), 2.67 (m, 1H), 2.89 (d, 1H, J=6.5 Hz), 2.97 (d,1H, J=6.9 Hz), 3.41 (m, 2H), 3.75 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H),4.42 (d, 1H, J=11.8 Hz), 4.65 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75(d, 1H, J=4.3 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.22 (m, 15H).

[0171] (2) In the same manner as described in Example 2-(7), thecompound prepared in (1) above (620 g, 0.44 mmol) was deprotected withzinc (722 mg, 11 mmol) and acylated with (R)-3-decanoyloxytetradecanoicacid (190 mg, 0.48 mmol) in the presence of EEDQ (170 mg, 0.58 mmol) toafford 254 mg (36%) of3-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-6-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0172] (3) In the same manner as described in Example 2-(8), thecompound prepared in (2) above (254 mg, 0.16 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 34 mg (13%) of3-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-6-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 169-171° C.; IR (film) 3306,2922, 2853, 1732, 1644, 1548, 1467, 1377, 1316, 1165, 1106, 1053, 856,722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.7 Hz), 1.1-1.7 (m,111H), 2.2-2.7 (m, 12H), 3.05 (m, 6H), 3.3-3.95 (m, 12H),4.11 (m, 1H),4.34 (m, 1H), 4.89 (m, 1H), 5.0-5.3 (m, 4H). ¹³C NMR(CDCl₃) 173.8,173.4, 171.1, 170.5, 101.3, 75.3, 74.9, 71.2, 71.0, 70.6, 68.8, 67.3,65.1, 61.4, 53.4, 50.7, 45.9, 41.5, 41.3, 39.6, 34.6, 32.0, 29.8, 29.6,29.4, 25.3, 25.1, 22.7, 14.1, 8.7.

[0173] Anal. Calcd. for C₈₇H₁₆₈N₃O₁₈P H₂O: C, 65.58; H, 10.75; N, 2.64;P, 1.94. Found: C, 65.60; H, 10.34; N, 2.36; P, 2.01.

EXAMPLE 8 (B7) Preparation of3-hydroxy-(S)-2-[(R)-3-nonanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₈H₁₇CO, X=Y=O, N=M=Q=0,R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0174] (1) In the same manner as described in Example 4-(5), thecompound prepared in Example 4-(4) (1.0 g, 1.56 mmol) was acylated with(R)-3-nonanoyloxytetradecanoic acid (660 mg, 1.71 mmol) in the presenceof EDC·MeI (560 mg, 1.87 mmol) and 4-pyrrolidinopyridine (50 mg) inCH₂Cl₂ (20 mL) to afford 1.31 g (83%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-nonanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 6H, J=6.8 Hz), 1.1-1.6(m, 32H), 1.37 (s, 3H), 1.46 (s, 3H), 2.27 (t, 2H, J=7.4 Hz), 2.50 (dd,1H, J=15.1, 6.0 Hz), 2.63 (dd, 1H, J=15.1, 6.8 Hz), 3.26 (m, 1H),3.35-4.0 (m, 9H), 4.32 (d, 1H, J=7.8 Hz), 4.41 (d, 1H, J=12.0 Hz), 4.51(m, 4H), 4.95 (m, 2H), 5.18 (m, 2H), 5.29 (d, 1H, J=17.2 Hz), 5.88 (m,1H), 7.36 (m, 5H).

[0175] (2) In the same manner as described in Example 4-(6) the compoundprepared in (1) above (1.29 g, 1.28 mmol) was deprotected in THF (20 mL)in the presence of dimethyl malonate (1.0 mL, 0.88 mmol) andtetrakis(triphenylphosphine)palladium(0) (200 mg) and then acylated with(R)-3-nonanoyloxytetradecanoic acid (540 mg, 1.41 mmol) in the presenceof EEDQ (370 mg, 1.5 mmol) to afford 1.02 g (65%) of3-benzyloxy-(S)-2-[(R)-3-nonanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-nonanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87(t, 12H, J=6.1 Hz),1.1-1.7 (m, 64H), 1.37 (s, 3H), 1.46 (s, 3H), 2.28 (m, 4H), 2.50 (dd,1H, J=15.5, 6.0 Hz), 2.62 (dd, 1H, J=14.8, 6.3 Hz), 3.27 (m, 2H), 3.44(m, 1H), 3.55 (m, 1H), 3.74 (m, 3H), 3.93 (m, 1H), 4.18 (m, 1H), 4.34(m, 2H), 4.57 (d, 1H, J=11.8 Hz), 4.65 (m, 2H), 4.97 (t, 1H, J=9.6 Hz),5.06 (d, 1H, J=8.6 Hz), 5.15 (m, 2H), 6.05 (d, 1H, J=8.2 Hz), 7.35 (m,5H).

[0176] (3) In the same manner as described in Example 4-(7) the compoundprepared in (2) above (1.0 g, 0.81 mmol) was deprotected in 90% aqueousAcOH (20 mL), treated with pyridine (0.080 mL, 0.98 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (215 mg, 0.89 mmol) inCH₂Cl₂ followed by diphenyl chlorophosphate (0.25 mL, 1.22 mmol),triethylamine (0.21 mL, 1.52 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 1.17 g (87%) of3-benzyloxy-(S)-2-[(R)-3-nonanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-nonanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.1Hz), 1.1-1.6 (m, 64H), 1.78 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H),2.18(m,3H),2.40(m, 2H), 2.67(m, 1H), 2.88 (d, 1H, J=6.5 Hz),2.97(d, 1H, J=6.9Hz), 3.41 (m, 2H), 3.72 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H), 4.42 (d,1H, J=11.8 Hz), 4.64 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75 (d, 1H,J=4.3 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.22 (m, 15H).

[0177] (4) In the same manner as described in Example 2-(7), thecompound prepared in (3) above (1.1 g, 0.66 mmol) was deprotected withzinc (2.2 g, 33 mmol) and acylated with (R)-3-nonanoyloxytetradecanoicacid (305 mg, 0.79 mmol) in the presence of EEDQ (235 mg, 0.95 mmol) toafford 373 mg (35%) of3-benzyloxy-(S)-2-[(R)-3-nonanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0178] (5) In the same manner as described in Example 2-(8), thecompound prepared in (4) above (373 mg, 0.23 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 43 mg (12%) of3-hydroxy-(S)-2-[(R)-3-nonanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 176-179° C.; IR (film) 3298,2956, 2923, 2853, 1733, 1646, 1551, 1467, 1337, 1316, 1254, 1166, 1106,1053, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.87 (t, 18H, J=6.7 Hz), 1.1-1.7(m, 105H), 2.2-2.7 (m, 12H), 3.03 (q, 6H, J=7.0 Hz), 3.3-4.3 (m, 14H),4.43 (d, 1H, J=7.1 Hz), 5.0-5.3 (m, 4H), 7.12 (d, 1H, J=7.7 Hz), 7.17(d, 1H, J=8.2 Hz); ¹³C NMR (CDCl₃) δ 173.9, 173.5, 173.3, 170.8, 170.5,170.1, 100.9, 75.5, 73.1, 71.4, 71.1, 70.9, 70.6, 67.8, 61.6, 60.7,54.3, 50.5, 45.8, 41.6, 41.4, 39.5, 34.6, 34.4, 32.0, 31.9, 29.8, 29.4,29.3, 25.4, 25.1, 22.7, 14.1, 8.6.

[0179] Anal. Calcd. for C₈₈H₁₆₄N₃O₁₈P: C, 65.81; H, 10.65; N, 2.74; P,2.02. Found: C, 66.14; H, 10.46; N, 2.58; P, 1.84.

EXAMPLE 9 (B8) Preparation of3-Hydroxy-(S)-2-[(R)-3-heptanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₆H₁₃CO, X=Y=O, N=M=Q=0,R₄=R₅=R₇=R₉=H, R₆=OH, P=1, R₈=PO₃H₂).

[0180] (1) In the same manner as described in Example 4-(5), thecompound prepared in Example 4-(4) (1.0 g, 1.56 mmol) was acylated with(R)-3-heptanoyloxytetradecanoic acid (610 mg, 1.71 mmol) in the presenceof EDC MeI (560 mg, 1.87 mmol) and 4-pyrrolidinopyridine (50 mg) inCH₂Cl₂ (20 mL) to afford 1.24 g (82%) of3-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-heptanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.0 Hz),1.1-1.6(m, 28H),1.38 (s, 3H), 1.47 (s, 3H), 2.29(t, 2H, J=7.4 Hz), 2.51 (dd,1H, J=15.1, 6.0 Hz), 2.63 (dd, 1H, J=15.1, 6.8 Hz), 3.26 (m, 1H),3.35-4.0 (m, 9H), 4.32 (d, 1H, J=7.3 Hz), 4.41 (d, 1H, J=12.0 Hz), 4.51(m, 4H), 4.95 (m, 2H), 5.18 (m, 2H), 5.29 (d, 1H, J=17.3 Hz), 5.88 (m,1H), 7.36 (m, 5H).

[0181] (2) In the same manner as described in Example 4-(6) the compoundprepared in (1) above (1.22 g, 1.25 mmol) was deprotected in THF (20 mL)in the presence of dimethyl malonate (1.0 mL, 0.88 mmol) andtetrakis(triphenylphosphine)palladium(0) (200 mg) and then acylated with(R)-3-heptanoyloxytetradecanoic acid (490 mg, 1.38 mmol) in the presenceof EEDQ (370 mg, 1.5 mmol) to afford 925 mg (62%) of3-benzyloxy-(S)-2-[(R)-3-heptanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-heptanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.7Hz), 1.1-1.7 (m, 56H), 1.37 (s, 3H), 1.46 (s, 3H), 2.32 (m, 4H), 2.50(dd, 1H, J=15.1, 6.0 Hz), 2.62 (dd, 1H, J=15.1, 6.8 Hz), 3.29 (m, 2H),3.44 (m, 1H), 3.55 (m, 1H), 3.74 (m, 3H), 3.93 (m, 1H), 4.18 (m, 1H),4.34 (m, 1H), 4.57 (d, 1H, J=11.8 Hz), 4.65 (m, 2H), 5.01 (m, 2H), 6.04(d, 1H, J=8.3 Hz), 7.36 (m, 5H).

[0182] (3) In the same manner as described in Example 4-(7) the compoundprepared in (2) above (920 mg, 0.76 mmol) was deprotected in 90% aqueousAcOH (20 mL), and then treated with pyridine (0.075 mL, 0.92 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (200 mg, 0.84 mmol) inCH₂Cl₂ followed by diphenyl chlorophosphate(0.24 mL, 1.14 mmol),triethylamine (0.21 mL, 1.52 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 1.03 g (83%) of3-benzyloxy-(S)-2-[(R)-3-heptanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-heptanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.87 (t, 12H, J=6.3Hz), 1.1-1.6 (m, 56H), 1.78 (s, 3H), 1.86 (s, 3H), 2.01 (m, 1H), 2.18(m, 3H), 2.40 (m, 2H), 2.67 (m, 1H), 2.88 (d, 1H, J=6.5 Hz), 2.97 (d,1H, J=6.9 Hz), 3.41 (m, 2H), 3.72 (m, 1H), 3.82 (m, 1H), 4.24 (m, 1H),4.42 (d, 1H, J=11.8 Hz), 4.64 (m, 3H), 5.16 (m, 1H), 5.39 (m, 2H), 5.75(d, 1H, J=4.3 Hz), 6.05 (d, 1H, J=8.4 Hz), 7.22 (m, 15H).

[0183] (4) In the same manner as described in Example 2-(7), thecompound prepared in (3) above (1.0 g, 0.61 mmol) was deprotected withzinc (2.0 g, 31 mmol) and acylated with (R)-3-heptanoyloxytetradecanoicacid (260 mg, 0.73 mmol) in the presence of EEDQ (220 mg, 0.88 mmol) toafford 203 mg (21%) of3-benzyloxy-(S)-2-[(R)-3-heptanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0184] (5) In the same manner as described in Example 2-(8), thecompound prepared in (4) above (203 mg, 0.13 mmol) was hydrogenated inthe presence of palladium hydroxide (100 mg) on carbon in EtOH (10 mL)and platinum oxide (200 mg) in EtOH/AcOH (10:1) to afford 39 mg (21%) of3-hydroxy-(S)-2-[(R)-3-heptanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 171-172° C.; IR (film) 3305,2955, 2924, 2853, 1734, 1644, 1553, 1466, 1377, 1170, 1102, 1052, 722cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.1-1.7 (m, 93H), 2.2-2.7(m, 12H), 3.06 (q, 6H, J=7.1 Hz), 3.3-4.0 (m, 13H), 4.23 (q, 1H, J=9.3Hz), 4.43 (d, 1H, J=8.2 Hz), 5.0-5.3 (m, 4H), 7.30 (d, 1H, J=8.5 Hz),7.43 (d, 1H, J=8.5 Hz); ¹³C NMR (CDCl₃) δ 173.8, 173.5, 173.2, 170.8,170.5, 170.2, 101.0, 77.2, 75.5, 73.1, 71.6, 71.1, 70.9, 70.6, 67.8,61.6, 60.8, 54.4, 50.5, 45.8, 41.6, 41.4, 39.5, 34.6, 34.4, 32.0, 31.6,29.8, 29.6, 29.4, 28.9, 25.4, 25.1, 22.7, 22.6, 14.1, 8.6.

[0185] Anal. Calcd. for C₇₈H₁₅₀N₃O₁₈P H₂O: C, 63.86; H, 10.44; N, 2.86;P, 2.11. Found: C, 63.47; H, 10.20; N, 2.59; P, 2.02.

EXAMPLE 10 (B9) Preparation of4-hydroxy-(S)-3-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O, N=P=1,M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, R₈=PO₃H₂).

[0186] (1) In the same manner as described in Example 4-(3) the compoundprepared in Example 4-(1) (3.1 g, 5.9 mmol) and(R)-3-(allyloxycarbonylamino)-4-benzyloxy-1-butanol (1.1 g, 3.94 mmol)were coupled in the presence of boron trifluoride etherate (3.0 mL, 23.6mmol) to afford 1.96 g (67%) of4-benzyloxy-(S)-3-(allyloxycarbonylamino)butyl2-deoxy-3,4,6-tri-O-acetyl-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid. In the same manner as described in Example 4-(4)the compound prepared above (1.8 g, 2.43 mmol) was deacylated inmethanol (25 mL) with ammonium hydroxide (5 mL) and then treated with2,2-dimethoxypropane (25 mL) and camphorsulfonic acid (100 mg) to afford1.34 g (84%) of 4-benzyloxy-(S)-3-(allyloxycarbonylamino)butyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside.

[0187] (2) In the same manner as described in Example 4-(5), thecompound prepared in (1) above (1.0 g, 1.53 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (670 mg, 1.68 mmol) in the presenceof EDC MeI (550 mg, 1.85 mmol) and 4-pyrrolidinopyridine (50 mg) inCH₂Cl₂ (15 mL) to afford 1.03 g (65%) of4-benzyloxy-(S)-3-(allyloxycarbonylamino)butyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.9 Hz),1.1-1.6(m, 34H),1.37 (s, 3H), 1.47 (s, 3H), 1.85 (m, 2H), 2.28 (t, 2H, J=7.6Hz), 2.50 (dd, 1H, J=15.1, 6.0 Hz), 2.63 (dd, 1H, J=15.1, 6.7 Hz), 3.30(m, 1H), 3.49 (m, 4H), 3.68 (t, 1H, J=9.4 Hz), 3.77 (t, 1H, J=10.4 Hz),3.92 (m, 3H), 4.54 (m, 5H), 4.69 (m, 2H), 5.1-5.4 (m, 4H), 5.91 (m, 1H),7.33 (m, 5H).

[0188] (3) In the same manner as described in Example 4-(6) the compoundprepared in (2) above (1.0 g, 0.97 mmol) was deprotected in THF (20 mL)in the presence of dimethyl malonate (1.0 mL, 0.88 mmol) andtetrakis(triphenylphosphine)palladium(0) (200 mg) and then acylated with(R)-3-decanoyloxytetradecanoic acid (425 mg, 1.07 mmol) in the presenceof EEDQ (317 mg, 1.28 mmol) to afford 660 mg (51%) of4-benzyloxy-(S)-3-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6Hz), 1.1-1.7 (m, 68H), 1.37 (s, 3H), 1.47 (s, 3H), 2.26 (q, 2H, J=7.1Hz), 2.41 (m, 2H), 2.62 (dd, 1H, J=14.9, 6.4 Hz), 3.29 (m, 1H), 3.48 (m,3H), 3.71 (m, 2H), 3.92 (m, 2H), 4.18 (m, 1H), 4.49 (m, 2H), 4.68 (q,2H, J=11.5 Hz), 5.15 (m, 2H), 5.55 (d, 1H, J=8.8 Hz), 6.17 (d, 1H, J=7.2Hz), 7.32(m, 5H).

[0189] (4) In the same manner as described in Example 4-(7) the compoundprepared in (3) above (640 mg, 0.48 mmol) was deprotected in 90% aqueousAcOH (20 mL), and then treated with pyridine (0.047 mL, 0.58 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (127 mg, 0.53 μmmol) inCH₂Cl₂ followed by diphenyl chlorophosphate (0.15 mL, 0.72 mmol),triethylamine (0.13 mL, 0.96 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 389 mg (47%) of4-benzyloxy-(S)-3-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-tichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6Hz), 1.1-1.6 (m, 68H), 1.79 (s, 3H), 1.86 (s, 3H), 2.22 (m, 4H), 2.40(m, 4H), 3.49 (m, 4H), 3.78 (m, 1H), 3.93 (m, 1H),4.1-4.5(m,5H),4.9-4.6(m, 4H),5.13(m, 2H),5.51 (t, 1H, J=8.9 Hz),5.84(d, 1H, J=6.9Hz), 6.09 (d, 1H, J=8.0 Hz), 7.26 (m, 15H).

[0190] (5) In the same manner as described in Example 2-(7), thecompound prepared in (4) above (375 g, 0.23 mmol) was deprotected withzinc (752 mg, 11.5 mmol) and acylated with(R)-3-decanoyloxytetradecanoic acid (101 mg, 0.25 mmol) in the presenceof EEDQ (70 mg, 0.28 mmol) to afford 270 mg (67%) of4-benzyloxy-(S)-3-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0191] (6) In the same manner as described in Example 2-(8), thecompound prepared in (5) above (270 mg, 0.15 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 93 mg (39%) of4-hydroxy-(S)-3-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-p-D-glucopyranosidetriethylammonium salt as a white powder: mp 179-181° C. (dec): IR (film)3287, 2956, 2923, 2853, 1734, 1654, 1552, 1466, 1378, 1246, 1164, 1106,1085, 1052, 721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.9 Hz),1.1-1.7 (m, 111H), 2.2-2.7 (m, 14H), 3.06 (q, 6H, J=6.9 Hz), 3.2-4.0 (m,13H), 4.21 (m, 1H), 4.50 (d, 1H, J=7.7 Hz), 5.0-5.3 (m, 4H),7.11 (m,2H); ¹³C NMR(CDCl₃) δ 173.8, 173.5, 173.3, 170.9, 170.5, 170.1,101.1,77.2, 75.5, 72.8, 71.3, 71.0, 70.6, 66.4, 64.0, 60.7, 54.8, 50.2,45.8, 41.6, 39.5, 34.6, 34.5, 34.4, 32.0, 30.6, 29.8, 29.7, 29.6, 29.5,29.4, 25.4, 25.1, 22.7, 14.2, 8.6.

[0192] Anal. Calcd. for C₈₈H₁₇₀N₃O₁₈P: C, 66.65; H, 10.78; N, 2.64; P,1.95. Found: C, 66.65; H, 10.68; N, 2.50; P, 1.94.

EXAMPLE 11 (B 10) Preparation of4-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O, N=M=Q=0,R₄=R₅=R₇=R₉=H, R₆=OH, P=2, R₈=PO₃H₂).

[0193] (1) In the same manner as described in Example 4-(3) the compoundprepared in Example 4-(1) (5.1 g, 9.7 mmol) and(R)-2-(allyloxycarbonylamino)-4-benzyloxy-1-butanol (1.8 g, 6.45 mmol)were coupled in the presence of boron trifluoride etherate (4.9 mL, 38.0mmol) to afford 2.92 g (61%) of4-benzyloxy-(S)-2-(allyloxycarbonylamino)propyl2-deoxy-3,4,6-tri-O-acetyl-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid. In the same manner as described in Example 4-(4)the compound prepared above (2.6 g, 3.51 mmol) was deacylated inmethanol (35 mL) with ammonium hydroxide (7 mL) and then treated with2,2-dimethoxypropane (35 mL) and camphorsulfonic acid (100 mg) to afford1.9 g (72%) of 4-benzyloxy-(S)-2-(allyloxycarbonylamino)butyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranoside.

[0194] (2) In the same manner as described in Example 4-(5), thecompound prepared in (1) above (1.0 g, 1.53 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (670 mg, 1.68 mmol) in the presenceof EDC MeI (550 mg, 1.85 mmol) and 4-pyrrolidinopyridine (50 mg) inCH₂Cl₂ (15 mL) to afford 1.28 g (81%) of4-benzyloxy-(S)-2-(allyloxycarbonylamino)butyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.9 Hz),1.1-1.7(m, 34H),1.37 (s, 3H), 1.47 (s, 3H), 1.82 (m, 2H), 2.28 (t, 2H, J=7.7Hz), 2.50 (dd, 1H, J=15.3, 6.0 Hz), 2.63 (dd, 1H, J=15.2, 6.7 Hz), 3.16(m, 1H), 3.56 (m, 3H), 3.65 (t, 1H, J=9.6 Hz), 3.75 (t, 1H, J=10.4 Hz),3.88 (m, 4H), 4.32 (d, 1H, J=8.5 Hz), 4.46 (s, 2H), 4.54 (m, 2H), 4.67(m, 2H), 4.90 (m, 1H), 5.26 (m, 3H), 5.89 (m, 1H), 7.33 (m, 5H).

[0195] (3) In the same manner as described in Example 4-(6) the compoundprepared in (2) above (1.25 g, 1.21 mmol) was deprotected in THF (20 mL)in the presence of dimethyl malonate (1.0 mL, 0.88 mmol) andtetrakis(triphenylphosphine)palladium(0) (200 mg) and then acylated with(R)-3-decanoyloxytetradecanoic acid (530 mg, 1.33 mmol) in the presenceof EEDQ (362 mg, 1.46 mmol) to afford 1.16 g (72%) of4-benzyloxy-(S)-3-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4,6-O-isopropylidene-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas a colorless amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.4Hz),1.1-1.7 (m, 68H), 1.37 (s, 3H), 1.45 (s, 3H), 2.26 (q, 2H, J=7.4Hz), 2.34 (m, 1H), 2.50 (dd, 1H, J=15.1, 6.0 Hz), 2.62 (dd, 1H, J=15.4,6.3 Hz), 3.12 (m, 1H), 3.5-3.95 (m, 7H), 4.14 (m, 1H), 4.29 (d, 1H,J=8.0 Hz), 4.67 (m, 2H), 4.86 (t, 1H, J=9.6 Hz),5.15 (m, 2H), 6.16 (d,1H, J=8.3 Hz), 7.35 (m, 5H).

[0196] (4) In the same manner as described in Example 4-(7) the compoundprepared in (3) above (1.1 g, 0.83 mmol) was deprotected in 90% aqueousAcOH (20 mL), and then treated with pyridine (0.080 mL, 1.0 mmol) and2,2,2-trichloro-1,1-dimethylethyl chloroformate (220 mg, 0.91 mmol) inCH₂Cl₂ followed by diphenyl chlorophosphate (0.26 mL, 1.25 mmol),triethylamine (0.23 mL, 1.66 mmol) and catalytic 4-pyrrolidinopyridine(50 mg) to afford 802 mg (56%) of4-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)—D-glucopyranosideas a colorless amorphous solid: ¹H NMR(CDCl₃) δ 0.87(t, 12H, J=6.8 Hz),1.1-1.6(m, 68H), 1.79 (s, 3H), 1.88 (s, 3H), 2.23 (m, 4H), 2.37 (m, 4H),3.57 (m, 4H), 3.83 (m, 1H), 4.29 (m, 3H), 4.44 (m, 2H), 4.69 (m, 4H),5.14 (m, 4H), 5.62 (d, 1H, J=7.6 Hz), 6.15 (d, 1H, J=8.3 Hz), 7.25 (m,15H).

[0197] (5) In the same manner as described in Example 2-(7), thecompound prepared in (4) above (750 mg, 0.43 mmol) was deprotected withzinc (1.42 g, 21.7 mmol) and acylated with(R)-3-decanoyloxytetradecanoic acid (190 mg, 0.48 mmol) in the presenceof EEDQ (130 mg, 0.53 mmol) to afford 483 mg (64%) of4-benzyloxy-(S)-2-[(R)-3-decanoyloxytetradecanoyl] butyl2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosideas a colorless amorphous solid.

[0198] (6) In the same manner as described in Example 2-(8), thecompound prepared in (5) above (483 mg, 0.27 mmol) was hydrogenated inthe presence of palladium hydroxide (150 mg) on carbon in EtOH (10 mL)and platinum oxide (300 mg) in EtOH/AcOH (10:1) to afford 238 mg (55%)of 4-hydroxy-(S)-2-[(R)-3-decanoyloxytetradecanoyl]butyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyltetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 181-183° C. (dec): IR (film)3294,2956, 2923, 2853, 1732, 1650, 1556, 1466, 1377, 1320, 1246, 1172,1108, 1082, 1058, 859, 721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H,J=6.9 Hz), 1.1-1.7 (m, 111H), 2.2-2.7 (m, 14H), 3.06(q, 6H, J=7.1 Hz),3.2-4.0 (m, 13H),4.21 (m, 1H), 4.46 (d, 1H, J=8.3 Hz),5.0-5.3 (m, 4H);¹³C NMR(CDCl₃) δ 173.9, 173.4, 173.2, 171.2, 170.7, 101.0, 77.2, 75.4,73.1, 71.4, 71.3, 71.1, 70.9, 70.6, 60.7, 58.4, 54.7, 46.3, 45.9, 41.6,41.1, 39.7, 34.8, 34.6, 34.4, 31.9, 29.8, 29.6, 29.5, 29.3, 25.4, 25.3,25.1, 22.7, 14.1, 8.6.

[0199] Anal. Calcd. for C₈₈H₁₇₀N₃O₁₈P: C, 66.51; H, 10.78; N, 2.64; P,1.95. Found: C, 66.81; H, 10.68; N, 2.53; P, 1.79.

EXAMPLE 12 (B11) Preparation ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0200] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (0.212 g, 1.08 mmol) was acylated with(R)-3-tetradecanoyloxytetradecanoic acid (0.541 g, 1.19 mmol) in thepresence of EDC-MeI (0.353 g, 1.19 mmol) to give 0.642 g (94%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-L-serine benzyl ester as a waxysolid: mp 56-61° C.; ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=˜7 Hz), 1.1-1.7 (m,42H), 2.29 (t, 2H, J=7.5 Hz), 2.50 (m, 2H), 3.87 (br t, 1H), 3.95 (m,2H), 4.65 (m, 1H), 5.1-5.25 (m, 3H), 6.69 (d, 1H, J=7 Hz), 7.34 (br s,5H).

[0201] (2) In the same manner as described in Example 2-(6), thecompound prepared in (1) above (0.19 g, 0.30 mmol) and the compoundprepared in Example 2-(4) (0.635 g, 0.478 mmol) were coupled in thepresence of mercury cyanide (0.3 g, 1.2 mmol) to give 0.425 g (77%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0202] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (0.405 g, 0.22 mmol) was deprotected withzinc (0.72 g, 11 mmol) and acylated with(R)-3-tetradecanoyloxytetradecanoic acid (0.12 g, 0.26 mmol) in thepresence of EEDQ (0.082 g, 0.33 mmol) to give 0.277 g (66%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 18H,J=˜6.5 Hz) 1.0-1.75 (m, 126H),2.15-2.45 (m, 10H),2.53 (dd, 1H, J=14.7,6.0 Hz),2.67 (dd, 1H, J=14, 6.0 Hz), 3.25 (br t, 1H, J=7 Hz), 3.35-3.75(m, 4H), 3.88 (dd, 1H, J=11.1 Hz), 4.23 dd, 1H, J=11.1, 3 Hz), 4.6-4.75(m, 2H), 5.03 (d, 1H, J=8.1 Hz), 5.05-5.25 (m, 4H), 5.48 (t, 1H, J=10Hz), 6.40 (d, 1H, J=7.5 Hz), 7.01 (d, 1H, J=8.1 Hz), 7.1-7.4 (m, 15H).

[0203] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (0.253 g, 0.133 mmol) was hydrogenated inthe presence of 5% palladium on carbon (50 mg) and platinum oxide (120mg) to give 0.155 g (62%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a colorless solid: mp 180° C. (dec); IR (film)3322, 2956, 2924, 2852, 1736, 1732, 1681, 1673, 1667, 1660, 1651, 1467,1456, 1247, 1174, 1110, 1081 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ0.88 (t, 18H,J=7 Hz),1.0-1.7 (m, 135H), 2.2-2.75 (m, 12H), 3.05 (q, 6H, J=7 Hz), 3.30(br s, 13H), 3.7-3.9 (m, 3H),3.96 (d, 1H, J=12 Hz), 4.05-4.3 (m, 2H),4.34 (m, 1H), 4.53 (d, 1H, J=7.8 Hz), 5.05-5.3 (m, 4H), 7.25-7.35 (m,2H); ¹³C NMR (CDCl₃) δ 173.4, 173.2, 171.0, 170.3, 170.2, 169.9, 169.8,100.8, 75.1, 73.4, 71.1, 70.7, 70.4, 70.3, 60.2, 54.3, 45.6, 41.2, 41.1,39.2, 34.6, 34.4, 34.2, 32.0, 29.8, 29.5, 25.4, 25.2, 22.7, 14.2, 8.6.

[0204] Anal. Calcd for C₉₉H₁₉₀N₃O₁₉P·5H₂O: C, 64.35; H, 10.91; N, 2.27;P, 1.68. Found: C, 64.16; H, 10.92; N, 2.37; P, 1.91.

EXAMPLE 13 (B12) Preparation ofN-[(R)-3-dodecanoyloxytetradecanoyl]-o-[2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₁H₂₃CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0205] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-dodecanoyloxytetradecanoic acid (935 mg, 2.2 mmol) in the presenceof EDC MeI (745 mg, 2.5 mmol) in CH₂Cl₂ to afford 1.08 g (90%) ofN-[(R)-3-dodecanoyloxytetradecanoyl]-L-serine benzyl ester: mp 53-54° C.¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.5 Hz), 1.1-1.6 (m, 46H), 2.30 (t, 2H,J=7.7 Hz), 2.50 (d, 2H, 5.6 Hz), 2.62 (t, 1H, J=6.2 Hz), 3.97 (m, 2H),4.65 (m, 1H), 5.19 (m, 3H), 6.63 (d, 1H, J=6.8 Hz), 7.35 (br s, 5H).

[0206] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-dodecanoyloxytetradecanoic acid (946 mg, 2.22 mmol) in thepresence of EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100mg) in CH₂Cl₂, and then deprotected in aqueous AcOH (25 mL) to afford1.30 g (81%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 28H), 1.59 (m, 4H), 2.30 (t, 2H, J=7.5 Hz), 2.52 (m, 2H), 3.42 (m,1H), 3.55 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.8, 4.6 Hz), 3.94 (m,2H), 4.57 (d, 1H, J=8.2 Hz), 4.71 (m, 2H), 5.07 (m, 2H), 5.27 (d, 1H,J=8.8 Hz).

[0207] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.30 g, 1.51 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (398 mg, 1.66 mmol) andpyridine (0.15 mL, 1.83 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.42 mL, 3.02 mmol), diphenyl chlorophosphate (0.47 mL,2.27 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.39 g (71%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.88 (m, 8H),1.1-1.7 (m, 46H), 1.77 (s, 3H), 1.85 (s, 3H), 2.23 (m, 6H), 3.34 (m,1H), 3.59 (m, 1H), 3.80 (m, 1H), 3.96 (m, 1H), 4.32 (m, 2H), 4.63 (m,2H), 4.83 (d, 1H, J=11.9 Hz), 5.02 (d, 1H, J=8.2 Hz), 5.20 (m, 1H), 5.65(m, 2H), 7.29 (m, 10H).

[0208] (4) The compound prepared in (3) above (1.30 g, 1.0 mmol) inCH₂Cl₂ (15 mL) was treated at 0° C. with TFA (5 mL) and then allowed towarm to room temperature for 18 h. The solvent was removed in vacuo andthe remaining TFA was removed by azeotroping with toluene. The lactolwas treated with the Vilsmeier reagent prepared from DMF (0.39 mL, 5.0mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) in CH₂Cl₂ (20 mL) at 0° C.The reaction was allowed to warm slowly to room temperature overnightand was partitioned between 50 mL of saturated aqueous NaHCO₃ and ether(50 mL). The layers were separated and the organic phase was dried overNa₂SO₄ and concentrated in vacuo. Purification by flash chromatographyon silica gel with 10% EtOAc/hexanes afforded 1.09 g (90%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.8 Hz),1.2-1.70 (m, 46H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.7 Hz),2.43 (m, 2H), 4.30 (m, 4H), 4.72 (m, 3 H), 5.09 (m, 1H), 5.50 (t, 1H,J=9.5 Hz), 5.79 (d, 1H, J=8.0 Hz), 6.27 (d, 1H, J=3.6 Hz), 7.19 (m,10H).

[0209] (5) To a solution of compounds prepared in (1) and (4) (540 mg,0.90 mmol, and 1.0 g, 0.82 mmol, respectively) in 1,2-dichloroethane (20mL), powdered 4A molecular sieves (300 mg) were added and the suspensionwas stirred for 30 min. AgOTf (1.16 g, 4.5 mmol) was added in oneportion, after 30 min the slurry was filtered through silica gel andeluted with 30% EtOAc/hexanes to afford 1.10 g (75%) ofN-[(R)-3-dodecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88(t, 12H, J=6.5 Hz), 1.1-1.65(m, 92H),1.77(s, 3H), 1.85(s, 3H),2.1-2.5(m, 8H), 3.67(m, 2H), 4.30 (m, 3H), 4.72(m, 5H), 5.18 (m, 4H), 5.46 (m, 1H), 6.07(m, 1H), 6.62 (d, 1H, J=7.9Hz), 7.05-7.45 (m, 15H).

[0210] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (1.0 g, 0.56 mmol) was deprotected withzinc (1.83 g, 28 mmol) and acylated with(R)-3-dodecanoyloxytetradecanoic acid (285 mg, 0.67 mmol) in thepresence of EEDQ (185 mg, 0.74 mmol) to afford 420 mg (44%) ofN-[(R)-3-dodecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0211] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (420 mg, 0.24 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon in EtOH (10 mL) andplatinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 240 mg (60%) ofN-[(R)-3-dodecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-dodecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 181-182° C.; IR (film) 3289,2956, 2920, 2851, 1731, 1656, 1557, 1467, 1378, 1182, 1108, 1080, 1052,852, 721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.7 Hz), 1.1-1.7(m, 123H), 2.2-2.7 (m, 12H), 3.06 (q, 6H, J=7.2 Hz), 3.35 (m, 1H), 3.70(m, 6H), 3.88 (m, 2H), 4.20 (m, 1H), 4.56 (d, 1H, J=8.1 Hz), 4.59 (br s,1H), 5.16 (m, 4H); ¹³C NMR (CDCl₃) δ 176.9,173.3, 173.2, 172.7, 169.6,169.1, 101.5, 74.8, 71.2, 70.9, 69.2, 60.5, 53.1, 51.4, 46.1, 41.5,41.0, 39.2, 34.3, 34.2, 34.0, 32.0, 29.8, 29.7, 29.4, 29.2, 25.6, 25.3,25.2, 25.1, 22.7, 14.1, 8.7.

[0212] Anal. Calcd. for C₉₃H₁₇₈N₃O₁₉P H₂O: C, 66.04; H, 10.73; N, 2.48;P, 1.83. Found: C, 66.04; H, 10.73; N, 2.48; P, 1.86.

EXAMPLE 14 (B13) Preparation ofN-[(R)-3-undecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₀H₂₁CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0213] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-undecanoyloxytetradecanoic acid (905 mg, 2.2 mmol) in the presenceof EDC MeI (745 mg, 2.5 mmol) in CH₂Cl₂ to afford 1.08 g (92%) ofN-[(R)-3-undecanoyloxytetradecanoyl]-L-serine benzyl ester: mp 53-54°C.; ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.9 Hz), 1.1-1.7 (m, 44H), 2.30 (t,2H, J=7.7 Hz), 2.49 (d, 2H, J=5.8 Hz), 3.99 (m, 2H), 4.65 (m, 1H), 5.19(m, 3H), 6.58 (d, 1H, J=6.9 Hz), 7.35 (br s, 5H).

[0214] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-undecanoyloxytetradecanoic acid (915 mg, 2.22 mmol) in thepresence of EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100mg) in CH₂Cl₂, and then deprotected in aqueous AcOH (25 mL) to afford1.41 g (82%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-undecanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 32H), 1.60 (m, 4H), 2.31 (t, 2H, J=7.5 Hz), 2.52 (m, 2H), 3.42 (m,1H), 3.55 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.8, 4.6 Hz), 3.94 (m,2H), 4.57 (d, 1H, J=8.2 Hz), 4.71 (m, 2H), 5.07 (m, 2H), 5.27 (d, 1H,J=8.7 Hz).

[0215] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.30, 1.53 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (403 mg, 1.68 mmol) andpyridine (0.15 mL, 1.85 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.43 mL, 3.06 mmol), diphenyl chlorophosphate (0.48 mL,2.30 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.37 g (70%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-undecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.88 (m, 8H),1.1-1.7 (m, 44H), 1.80 (s, 3H), 1.89 (s, 3H), 2.23 (m, 6H), 3.58 (m,3H), 4.32 (m, 1H), 4.71 (m, 2H), 4.83 (d, 1H, J=12.1 Hz), 5.01 (d, 1H,J=8.1 Hz), 5.20 (m, 1H), 5.62 (m, 2H), 7.25 (m, 10H).

[0216] (4) In the same manner as described in Example 13-(4), thecompound prepared in (4) above (1.28 g, 1.0 mmol) was deprotected withTFA (5 mL) and then treated with the Vilsmeier reagent generated fromDMF (0.39 mL, 5.0 mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) to give1.12 g (93%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-undecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.7 Hz),1.1-1.55 (m, 44H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (m, 2H), 2.43 (m,2H), 4.34 (m, 4H), 4.72 (m, 3H), 5.09 (m, 1H), 5.50 (t, 1H, J=9.6 Hz),5.80 (d, 1H, J=8.0 Hz), 6.26 (d, 1H, J=3.4 Hz), 7.26 (m, 10H).

[0217] (5) In the same manner as described in Example 13-(5), compoundsprepared in (1) and (4) above (530 mg, 0.90 mmol, and 1.0 g, 0.83 mmol,respectively) were coupled in the presence of AgOTf (1.16 g, 4.5 mmol)to afford 1.11 g (76%) ofN-[(R)-3-undecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-undecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (m, 12H), 1.0-1.65 (m, 88H), 1.77(s, 3H), 1.85 (s, 3H), 2.1-2.5 (m, 8H), 3.37 (m, 1H), 3.64 (m, 1H), 3.85(m, 1H), 4.30 (m, 3H), 4.78 (m, 5H), 5.18 (m, 4H), 5.46 (m, 1H), 6.07(m, 1H), 6.62 (d, 1H, J=7.7 Hz), 7.05-7.45 (m, 15H).

[0218] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (1.0 g, 0.57 mmol) was deprotected withzinc (2.0 g, 30.5 mmol) and acylated with(R)-3-undecanoyloxytetradecanoic acid (280 mg, 0.68 mmol) in thepresence of EEDQ (185 mg, 0.75 mmol) to afford 470 mg (50%) ofN-[(R)-3-undecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0219] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (470 mg, 0.27 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon in EtOH (10 mL) andplatinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 130 mg (30%) ofN-[(R)-3-undecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-undecanoyloxytetradecanoylamino]-3-O-[(R)-3-undecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 181-183° C.; IR (film) 3294,2923, 2853, 1734, 1655, 1466, 1377, 1163, 1080, 721 cm⁻¹; ¹HNMR(CDCl₃—CD₃OD) δ 0.88(t, 18H, J=6.8 Hz), 1.1-1.7(m, 117H),2.2-2.7(m,12H),3.06(q, 6H, J=7.1 Hz), 3.4-3.2 (m, 5H), 3.6-3.9 (m, 4H), 4.20 (d,1H, 9.8 Hz), 4.54 (d, 1H, J=8.0 Hz), 4.62 (br. s, 1H), 5.17 (m, 4H); ¹³CNMR (CDCl₃) δ 173.5, 173.3, 172.8, 172.2, 169.6, 169.1, 101.5, 77.2,74.8, 70.9, 69.2, 60.5, 58.5, 53.1, 51.5, 46.1, 41.5, 41.1, 39.2, 34.6,34.4, 34.1, 32.0, 29.8, 29.7, 29.4, 29.2, 25.6, 25.2, 25.1, 22.7, 18.5,14.2, 8.7.

[0220] Anal. Calcd. for C₉₀H₁₇₂N₃O₁₉P: C, 66.26; H, 10.63; N, 2.58; P,1.90. Found: C, 66.56; H, 10.57; N, 2.47; P, 1.91.

EXAMPLE 15 (B14) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-D-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈═PO₃H₂).

[0221] (1) In the same manner as described in Example 2-(5), D-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (875 mg, 2.2 mmol) in the presenceof EDC MeI (745 mg, 2.5 mmol) in CH₂Cl₂ to afford 1.05 g (91%) ofN-[(R)-3-decanoyloxytetradecanoyl]-D-serine benzyl ester: mp 51-52° C.;¹H NMR (CDCl₃) δ 0.88 (m, 6H), 1.1-1.7 (m, 34H), 2.30 (t, 2H, J=7.7 Hz),2.50 (m, 2H),3.68 (s, 1H), 3.93 (d, 2H, J=3.1 Hz), 4.62 (m, 1H), 5.22(m, 3H), 6.63 (d, 1H, J=6.9 Hz), 7.35 (br s, 5H).

[0222] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (884 mg, 2.22 mmol) in the presenceof EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100 mg) inCH₂Cl₂, and then deprotected in aqueous AcOH (25 mL) to afford 1.30 g(77%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 30H), 1.59 (m, 4H), 2.30 (t, 2H, J=7.5 Hz), 2.52 (m, 2H), 3.42 (m,1H), 3.55 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.8, 4.6 Hz), 3.94 (m,2H),4.57 (d, 1H, J=8.2 Hz),4.71 (m, 2H), 5.07 (m, 2H), 5.27 (d, 1H,J=8.8 Hz).

[0223] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.25 g, 1.50 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (396 mg, 1.65 mmol) andpyridine (0.15 mL, 1.81 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.42 mL, 3.00 mmol), diphenyl chlorophosphate (0.47 mL,2.25 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.31 g (69%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.89 (m, 8H),1.1-1.7 (m, 34H), 1.82 (s, 3H), 1.90 (s, 3H), 2.30 (m, 4H), 3.40 (q, 1H,J=9.6 Hz), 3.65 (m, 1H), 3.89 (m, 1H), 4.32 (m, 2H), 4.63 (m, 2H), 4.82(d, 1H, J=12.1 Hz), 5.01 (d, 1H, J=8.2 Hz), 5.63 (m, 2H), 7.29 (m, 10H).

[0224] (4) In the same manner as described in Example 13-(4), thecompound prepared in (3) above (1.27 g, 1.0 mmol) was deprotected withTFA (5 mL) and then treated with the Vilsmeier reagent generated fromDMF (0.39 mL, 5.0 mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) to give1.06 g (89%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.6 Hz),1.1-1.55 (m, 34H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.7 Hz),2.43 (m, 2H), 4.32 (m, 4H), 4.71 (m, 3H), 4.83 (m, 3H), 5.09 (m, 1H),5.50 (t, 1H, J=9.5 Hz), 5.77 (d, 1H, J=8.0 Hz), 6.26 (d, 1H, J=3.4 Hz),7.20 (m, 10H).

[0225] (5) In the same manner as described in Example 13-(5), compoundsprepared in (1) and (4) above above (520 mg, 0.90 mmol, and 1.0 g, 0.84mmol, respectively) were coupled in the presence of AgOTf (1.16 g, 4.5mmol) to afford 1.13 g (78%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-D-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6 Hz), 1.1-1.65 (m,68H), 1.82 (s, 3H), 1.89 (s, 3H), 2.2-2.6 (m, 8H), 3.40(m, 1H), 3.64 (m,1H), 4.01 (m, 2H),4.27(m, 2H),4.44(d, 1H, J=7.1 Hz), 4.60 (m, 2H), 4.77(m, 2H), 5.19 (m, 6H), 6.61 (d, 1H, J=8.3 Hz), 7.05-7.45 (m, 15H).

[0226] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (1.0 g, 0.58 mmol) was deprotected withzinc (1.9 g, 29 mmol) and acylated with (R)-3-decanoyloxytetradecanoicacid (280 mg, 0.70 mmol) in the presence of EEDQ (190 mg, 0.77 mmol) toafford 420 mg (44%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-D-serinebenzyl ester as an amorphous solid.

[0227] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (420 mg, 0.25 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon in EtOH (10 mL) andplatinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 118 mg (30%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-D-serinetriethylammonium salt as a white powder: mp 179-181° C.; IR (film) 3283,3100, 2921, 2852, 1732, 1660, 1651, 1564, 1556, 1464, 1417, 1378, 1322,1181, 1061, 856, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.8Hz), 1.1-1.7 (m, 111H), 2.2-2.7 (m, 12H), 3.06 (m, 6H), 3.33 (m, 5H),3.78 (m, 2H), 3.95 (m, 2H), 4.22 (m, 1H), 4.45 (d, 1H, J=7.5 Hz), 4.68(br. s, 1H), 5.13 (m, 3H), 5.26 (m, 1H); ¹³C NMR (CDCl₃) δδ 173.7,173.5, 173.1, 171.1, 169.9, 100.3, 75.1, 73.9, 71.9, 71.1, 70.9, 70.2,60.9, 53.9, 52.7, 46.0, 41.3, 40.8, 39.4, 34.6, 34.4, 31.9, 29.8, 29.7,29.5, 29.4, 25.6, 25.4, 25.2, 25.1, 22.7, 14.1, 8.6.

[0228] Anal. Calcd. for C₈₇H₁₆₆N₃O₁₉P: C, 65.75; H, 10.53; N, 2.64; P,1.95. Found: C, 65.32; H, 10.28; N, 2.53; P, 1.89.

EXAMPLE 16 (B15) Preparation of ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₉=PO₃H₂).

[0229] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (250 mg, 1.08 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (478 mg, 1.2 mmol) in the presenceof EDC MeI (357 mg, 1.2 mmol) in CH₂Cl₂ to afford 0.52 g (84%) ofN-[(R)-3-heptanoyloxytetradecanoyl]-L-serine benzyl ester: mp 52-53° C.;¹H NMR (CDCl₃) δ 0.87 (t, 6H, J=6.9 Hz), 1.1-1.7 (m, 34H), 2.29 (t, 2H,J=7.5 Hz), 2.49 (d, 2H, J=5.8 Hz), 3.67 (s, 1H),3.97 (m, 2H), 4.63 (m,1H), 5.19 (m, 3H), 6.61 (d, 1H, J=7.1 Hz), 7.35 (br s, 5H).

[0230] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (500 mg, 0.87 mmol), and the compoundprepared in Example 15-(4) (1.08 g, 0.90 mmol) were coupled in thepresence of AgOTf (1.16 g, 4.5 mmol) to afford 1.35 g (89%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-0-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6 Hz), 1.0-1.65 (m,68H), 1.77 (s, 3H), 1.85 (s, 3H), 2.1-2.5 (m, 8H), 3.38 (q, 1H, J=9.1Hz), 3.65 (m, 1H), 3.84 (m, 1H),4.27 (m, 3H),4.70 (m, 5H),4.84 (m, 4H),5.14 (m, 3H), 5.46 (t, 1H, J=9.7 Hz), 6.07 (m, 1H), 6.62 (d, 1H, J=8.0Hz), 7.05-7.45 (m, 15H).

[0231] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (600 mg, 0.34 mmol) was deprotected withzinc (1.13 g, 17.2 mmol) and acylated with(R)-3-decanoyloxytetradecanoic acid (150 mg, 0.38 mmol) in the presenceof EEDQ (124 mg, 0.50 mmol) to afford 362 mg (60%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0232] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (300 mg, 0.17 mmol) was hydrogenated inthe presence of palladium on carbon (100 mg) and platinum oxide (200 mg)in THF/AcOH (10:1) to afford 120 mg (44%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 175-176° C.; IR (film) 3304,2956, 2923, 2853, 1733, 1654, 1541, 1466, 1377, 1164, 1107, 1080, 845,721 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.9 Hz), 1.1-1.7 (m,111H), 2.2-2.75 (m, 12H), 3.07 (q, 6H, J=7.2 Hz), 3.37 (m, 1H), 3.5-3.95(m, 8H), 4.21 (q, 1H, 11.0 Hz), 4.54 (d, 1H, J=8.9 Hz),4.61 (br. s,1H),5.17(m, 4H),7.10(d, 1H, J=9.0 Hz),7.43(d, 1H, J=7.9 Hz); ¹³C NMR(CDCl₃) δ 176.3, 173.4, 173.2, 172.8, 172.0, 169.6, 169.2, 101.4, 74.7,70.9, 69.3, 60.4, 53.2, 51.6, 46.1, 41.4, 41.0, 39.1, 34.5, 34.3, 34.2,34.1, 31.9, 29.8, 29.7, 29.6, 29.4, 29.3, 29.2, 25.5, 25.1, 25.0, 22.7,14.1, 8.6.

[0233] Anal. Calcd. for C₈₇H₁₆₆N₃O₁₉P·H₂O: C, 65.01; H, 10.54; N, 2.61;P, 1.93. Found: C, 64.92; H, 10.38; N, 2.58; P, 2.06.

EXAMPLE 17 (B16) Preparation ofN-[(R)-3-nonanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₂=R₃=N—C₈H₁₇CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0234] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-nonanoyloxytetradecanoic acid (780 mg, 2.2 mmol) in the presenceof EDC MeI (845 mg, 2.5 mmol) in CH₂Cl₂ to afford 1.0 g (89%) ofN-[(R)-3-nonanoyloxytetradecanoyl]-L-serine benzyl ester: mp 52-53° C.;¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.6 Hz), 1.1-1.7 (m, 32H), 2.30 (t, 2H,J=7.7 Hz), 2.51 (d, 2H, J=5.8 Hz), 2.62 (t, 1H, J=6.0 Hz), 3.98 (m, 2H),4.65 (m, 1H), 5.19 (m, 3H), 6.58 (d, 1H, J=6.8 Hz), 7.35 (br s, 5H).

[0235] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-nonanoyloxytetradecanoic acid (852 mg, 2.22 mmol) in the presenceof EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100 mg) inCH₂Cl₂, and then deprotected in aqueous AcOH (25 mL) to afford 1.31 g(79%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-nonanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-p-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 28H), 1.59 (m, 4H), 2.30 (t, 2H, J=7.5 Hz), 2.52 (m, 2H), 3.42 (m,1H), 3.55 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.8, 4.6 Hz), 3.94 (m,2H), 4.57 (d, 1H, J=8.2 Hz), 4.71 (m, 2H), 5.07 (m, 2H), 5.27 (d, 1H,J=8.8 Hz).

[0236] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.25 g, 1.52 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (400 mg, 1.67 mmol) andpyridine (0.15 mL, 1.84 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.42 mL, 3.04 mmol), diphenyl chlorophosphate (0.47 mL,2.28 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.30 g (67%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-nonanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)—D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.88 (m, 8H),1.1-1.7 (m, 32H), 1.82 (s, 3H), 1.89 (s, 3H), 2.22 (m, 6H), 3.33 (m,1H), 3.53 (m, 1H), 3.80 (m, 1H), 3.96 (m, 1H), 4.31 (m, 2H), 4.55 (m,2H), 4.83 (d, 1H, J=12.0 Hz), 5.01 (d, 1H, J=7.9 Hz), 5.62 (m, 1H), 7.28(m, 10H).

[0237] (4) In the same manner as described in Example 13-(4), thecompound prepared in (3) above (1.26 g, 1.0 mmol) was deprotected withTFA (5 mL) and then treated with the Vilsmeier reagent generated fromDMF (0.39 mL, 5.0 mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) to give1.07 g (91%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-nonanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.9 Hz),1.25-1.55 (m, 32H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.7 Hz),2.43 (m, 2H), 4.34 (m, 4H), 4.70 (m, 3H), 4.83 (m, 3H), 5.09 (m, 1H),5.51 (t, 1H, J=10.2 Hz), 5.78 (d, 1H, J=8.0 Hz), 6.25 (d, 1H, J=3.6 Hz),7.19 (m, 10H).

[0238] (5) In the same manner as described in Example 13-(5), compoundsprepared in (1) and (4) above (505 mg, 0.90 mmol, and 1.0 g, 0.85 mmol,respectively) were coupled in the presence of AgOTf (1.16 g, 4.5 mmol)to afford 1.03 g (71%) ofN-[(R)-3-nonanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-nonanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.9 Hz), 1.0-1.65 (m,64H), 1.78 (s, 3H), 1.82 (s, 3H), 2.1-2.5 (m, 8H), 3.38 (m, 1H), 3.64(m, 1H), 3.83 (m, 1H), 4.25 (m, 3H), 4.73 (m, 5H), 5.18 (m, 5H), 6.07(m, 1H), 6.60 (d, 1H, J=7.8 Hz), 7.05-7.45 (m, 15H).

[0239] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (1.0 g, 0.59 mmol) was deprotected withzinc (1.93 g, 29.5 mmol) and acylated with(R)-3-nonanoyloxytetradecanoic acid (273 mg, 0.71 mmol) in the presenceof EEDQ (195 mg, 0.78 mmol) to afford 405 mg (42%) ofN-[(R)-3-nonanoyloxytetradecanoyl]-O-[deoxy-4-O-diphenylphosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0240] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (405 mg, 0.25 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon in EtOH (10 mL) andplatinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 185 mg (48%) ofN-[(R)-3-nonanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-nonanoyloxytetradecanoylamino]-3-O-[(R)-3-nonanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 177-179° C.; IR (film) 3306,2955, 2923, 2853, 1732, 1660, 1538, 1467, 1378, 1252, 1165, 1106, 1080,960, 844,722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.8 Hz),1.1-1.7 (m, 105H), 2.2-2.75 (m, 12H), 3.07 (q, 6H, J=7.1 Hz), 3.2-3.5(m, 5H), 3.85 (m, 4H), 4.23 (d, 1H, 10.2 Hz), 4.51 (d, 1H, J=8.0 Hz),4.64 (br. s, 1H), 5.18 (m, 4H); ¹³C NMR (CDCl₃) δ 173.3, 172.8, 172.2,169.6, 169.1, 101.5, 74.8, 70.9, 70.8, 69.3, 60.5, 53.2, 51.5, 46.1,41.5, 41.0, 39.2, 34.5, 34.3, 34.1, 32.0, 31.9, 29.8, 29.6, 29.4, 29.3,25.6, 25.2, 25.1, 22.7, 14.1, 8.7.

[0241] Anal. Calcd. for C₈₄H₁₆₀N₃O₁₉P: C, 65.21; H, 10.42; N, 2.72; P,2.00. Found: C, 65.48; H, 10.32; N, 2.62; P, 2.12.

EXAMPLE 18 (B17) Preparation ofN-[(R)-3-octanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-octanoyloxytetradecanoylamino]-3-O-[(R)-3-octanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₇H₁₅CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0242] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-octanoyloxytetradecanoic acid (815 mg, 2.2 mmol) in the presenceof EDC MeI (745 mg, 2.5 mmol) in CH₂Cl₂ to afford 1.02 g (93%) ofN-[(R)-3-octanoyloxytetradecanoyl]-L-serine benzyl ester: mp 50-51° C.;¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.8 Hz), 1.1-1.7 (m, 30H), 2.30 (t, 2H,J=7.7 Hz), 2.51 (d, 2H, J=5.8 Hz), 2.60 (t, 1H, J=6.0 Hz), 3.97 (m, 2H),4.65 (m, 1H), 5.22 (m, 3H), 6.61 (d, 1H, J=6.9 Hz), 7.35 (br s, 5H).

[0243] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-octanoyloxytetradecanoic acid (821 mg, 2.22 mmol) in the presenceof EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100 mg) inCH₂Cl₂, and then deprotected in 90% aqueous AcOH (25 mL) to afford 1.35g (83%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-octanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 26H), 1.60 (m, 4H), 2.30 (t, 2H, J=7.5 Hz), 2.53 (m, 2H), 3.42 (m,1H), 3.53 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.8, 4.4 Hz), 3.94 (m,2H), 4.56 (d, 1H, J=8.3 Hz), 4.64 (d, 1H, J=11.8 Hz), 4.77 (d, 1H,J=11.8 Hz), 5.08 (m, 2H), 5.30 (br. s, 1H).

[0244] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.30 g, 1.61 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (425 mg, 1.77 mmol) andpyridine (0.16 mL, 1.95 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.45 mL, 3.22 mmol), diphenyl chlorophosphate (0.50 mL,2.42 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.42 g (71%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-octanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.88 (m, 8H),1.1-1.7 (m, 30H), 1.82 (s, 3H), 1.89 (s, 3H), 2.23 (m, 6H), 3.37 (m,1H), 3.65 (m, 1H), 3.83 (m, 1H), 3.96 (m, 1H), 4.55 (m, 2H), 4.83 (d,1H, J=11.8 Hz), 5.01 (d, 1H, J=8.2 Hz), 5.20 (m, 1H), 7.29 (m, 10H).

[0245] (4) In the same manner as described in Example 13-(4), thecompound prepared in (3) above (1.24 g, 1.0 mmol) was deprotected withTFA (5 mL) and then treated with the Vilsmeier reagent generated fromDMF (0.39 mL, 5.0 mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) to give1.0 g (87%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-octanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.7 Hz),1.25-1.55 (m, 30H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.7 Hz),2.43 (m, 2H), 4.29 (m, 4H), 4.72 (m, 3H), 5.09 (m, 1H), 5.51 (t, 1H,J=9.9 Hz), 5.79 (d, 1H, J=7.9 Hz), 6.25 (d, 1H, J=3.5 Hz), 7.29 (m,10H).

[0246] (5) In the same manner as described in Example 13-(5), compoundsprepared in (1) and (4) above (490 mg, 0.90 mmol, and 1.0 g, 0.86 mmol,respectively) were coupled in the presence of AgOTf (1.16 g, 4.5 mmol)to afford 0.99 g (69%) ofN-[(R)-3-octanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-octanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.9 Hz), 1.0-1.65 (m,60H), 1.77 (s, 3H), 1.85 (s, 3H), 2.1-2.5 (m, 8H), 3.37 (m, 1H), 3.65(m, 1H), 3.83 (m, 1H), 4.27 (m, 3H), 4.72 (m, 5H), 5.18 (m, 4H), 5.46(t, 1H, J=9.8 Hz), 6.06 (m, 1H), 6.60 (d, 1H, J=8.0 Hz), 7.05-7.45 (m,15H).

[0247] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (0.95 g, 0.57 mmol) was deprotected withzinc (1.86 g, 28.5 mmol) and acylated with(R)-3-octanoyloxytetradecanoic acid (252 mg, 0.68 mmol) in the presenceof EEDQ (185 mg, 0.75 mmol) to afford 433 mg (47%) ofN-[(R)-3-octanoyloxytetradecanoyl]—O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-octanoyloxytetradecanoylamino]-3-O-[(R)-3-octanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0248] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (433 mg, 0.27 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon (250 mg) in EtOH (10 mL)and platinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 196 mg (48%)ofN-[(R)-3-octanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-octanoyloxytetradecanoylamino]-3-O-[(R)-3-octanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 177-178° C.; IR (film) 3296,2956, 2923, 2853, 1732, 1645, 1546, 1466, 1378, 1315, 1170, 1082, 1056,961, 846, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD)60.88(t, 18H, J=6.6 Hz), 1.1-1.7(m, 99H), 2.2-2.75 (m, 12H), 3.08 (q, 6H, J=7.1 Hz), 3.39 (d, 1H, J=8.8Hz), 3.6-4.0 (m, 8H), 4.22 (q, 1H, 10.3 Hz), 4.53 (d, 1H, J=8.2 Hz),4.63 (m, 1H), 5.18 (m, 4H), 7.04 (d, 1H, J=8.8 Hz), 7.42 (d, 1H, J=8.0Hz); ¹³C NMR (CDCl₃) 8176.8, 173.3, 173.2, 172.7, 172.2, 169.6, 169.1,101.5, 74.8, 70.9, 70.8, 69.3, 60.5, 53.2, 51.5, 46.2, 41.5, 41.1, 39.2,34.5, 34.3, 34.1, 34.0, 32.0, 31.8, 29.8, 29.6, 29.4, 29.3, 29.2, 29.1,25.6, 25.3, 25.2, 25.0, 22.7, 14.1, 8.7.

[0249] Anal. Calcd. for C₈₁H₁₅₄N₃O₁₉P H₂O: C, 63.87; H, 10.32; N, 2.76;P, 2.03. Found: C, 63.96; H, 10.29; N, 2.69; P, 1.67.

EXAMPLE 19 (B18) Preparation ofN-[(R)-3-heptanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serineTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₆H₁₃CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0250] (1) In the same manner as described in Example 2-(5), L-serinebenzyl ester (390 mg, 2.0 mmol) was acylated with(R)-3-heptanoyloxytetradecanoic acid (780 mg, 2.2 mmol) in the presenceof EDC MeI (745 mg, 2.5 mmol) in CH₂Cl₂ to afford 0.97 g (91%) ofN-[(R)-3-heptanoyloxytetradecanoyl]-L-serine benzyl ester: mp 46-48° C.;¹H NMR(CDCl₃) δ 0.88(t, 6H, J=6.9 Hz), 1.1-1.7(m, 28H),2.30(t, 2H, J=7.7Hz),2.50(d, 2H, J=5.8 Hz), 2.62 (t, 1H, J=6.0 Hz), 3.97 (m, 2H), 4.65(m, 1H), 5.19 (m, 3H), 6.61 (d, 1H, J=6.9 Hz), 7.35 (br s, 5H).

[0251] (2) In the same manner as described in Example 2-(2), thecompound prepared in Example 2-(1) (1.0 g, 2.02 mmol) was acylated with(R)-3-heptanoyloxytetradecanoic acid (790 mg, 2.22 mmol) in the presenceof EDC MeI (720 mg, 2.4 mmol) and 4-pyrrolidinopyridine (100 mg) inCH₂Cl₂, and then deprotected in 90% aqueous AcOH (25 mL) to afford 1.30g (81%) of 2-(trimethylsilyl)ethyl2-deoxy-3-O-[(R)-3-heptanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.00 (s, 9H), 0.88 (m, 8H), 1.25(m, 24H), 1.59 (m, 4H), 2.30 (t, 2H, J=7.5 Hz), 2.52 (m, 2H), 3.42 (m,1H), 3.55 (m, 1H), 3.66 (m, 1H), 3.83 (dd, 1H, J=11.5, 4.2 Hz), 3.94 (m,2H), 4.57 (d, 1H, J=8.3 Hz), 4.64 (d, 1H, J=12.1 Hz), 4.76 (d, 1H,J=11.9 Hz), 5.09 (m, 2H), 5.31 (d, 1H, J=8.7 Hz).

[0252] (3) In the same manner as described in Example 2-(3), thecompound prepared in (2) above (1.25 g, 1.58 mmol) was treated with2,2,2-trichloro-1,1-dimethylethyl chloroformate (417 mg, 1.74 mmol) andpyridine (0.15 mL, 1.91 mmol) in CH₂Cl₂ (25 mL) followed bytriethylamine (0.44 mL, 3.16 mmol), diphenyl chlorophosphate (0.49 mL,2.37 mmol) and 4-pyrrolidinopyridine (100 mg) to afford 1.34 g (69%) of2-(trimethylsilyl)ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-heptanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.0 (s, 9H), 0.88 (m, 8H),1.1-1.7 (m, 28H), 1.82 (s, 3H), 1.89 (s, 3H), 2.35 (m, 4H), 3.37 (m,1H), 3.61 (m, 1H), 3.80 (m, 1H), 4.32 (m, 2H), 4.63 (m, 2H), 4.83 (d,1H, J=12.0 Hz), 5.01 (d, 1H, J=8.2 Hz), 5.62 (m, 2H), 7.29 (m, 10H).

[0253] (4) In the same manner as described in Example 13-(4), thecompound prepared in (3) above (1.23 g, 1.0 mmol) was deprotected withTFA (5 mL) and then treated with the Vilsmeier reagent generated fromDMF (0.39 mL, 5.0 mmol) and oxalyl chloride (0.22 mL, 2.5 mmol) to give1.0 g (87%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-heptanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.9 Hz),1.25-1.55 (m, 28H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.6 Hz),2.43 (m, 2H), 4.26 (m, 4H), 4.73 (m, 3H), 5.09 (m, 1H), 5.51 (t, 1H,J=10.2 Hz), 5.77 (d, 1H, J=8.0 Hz), 6.25 (d, 1H, J=3.3 Hz), 7.19 (m,10H).

[0254] (5) In the same manner as described in Example 13-(5), compoundsprepared in (1) and (4) above (480 mg, 0.90 mmol, and 0.98 g, 0.86 mmol,respectively) were coupled in the presence of AgOTf (1.16 g, 4.5 mmol)to afford 1.06 g (75%) ofN-[(R)-3-heptanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-heptanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (m, 12H), 1.0-1.65 (m, 56H), 1.77(s,3H), 1.85 (s, 3H), 2.1-2.5 (m, 8H), 3.38 (m, 1H), 3.64 (m, 1H), 3.83 (m,1H), 4.25 (m, 3H), 4.78 (m, 5H), 5.16 (m, 4H), 5.46 (t, 1H, J=9.9 Hz),6.06 (m, 1H), 6.60 (d, 1H, J=7.7 Hz), 7.05-7.45 (m, 15H).

[0255] (6) In the same manner as described in Example 2-(7), thecompound prepared in (5) above (1.0 g, 0.61 mmol) was deprotected withzinc (2.0 g, 30.5 mmol) and acylated with(R)-3-heptanoyloxytetradecanoic acid (260 mg, 0.73 mmol) in the presenceof EEDQ (200 mg, 0.80 mmol) to afford 440 mg (45%) ofN-[(R)-3-heptanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinebenzyl ester as an amorphous solid.

[0256] (7) In the same manner as described in Example 2-(8), thecompound prepared in (6) above (440 mg, 0.28 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon (250 mg) in EtOH (10 mL)and platinum oxide (400 mg) in EtOH/AcOH (10:1) to afford 208 mg (51%)ofN-[(R)-3-heptanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-heptanoyloxytetradecanoylamino]-3-O-[(R)-3-heptanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinetriethylammonium salt as a white powder: mp 176-177° C.; IR (film) 3307,2956, 2924, 2854, 1732, 1650, 1545, 1466, 1378, 1316, 1170, 1080, 956,841, 722 cm^(−1;) ¹H NMR(CDCl₃—CD₃OD)60.88(m, 18H), 1.1-1.7 (m, 93H),2.2-2.75 (m, 12H), 3.08 (q, 6H, J=7.2 Hz), 3.40 (d, 1H, J=10.2 Hz),3.6-4.0 (m, 7H), 4.24 (m, 2H), 4.52 (d, 1H, J=8.0 Hz), 4.63 (m, 1H),5.19 (m, 4H), 7.04 (d, 1H, J=8.6 Hz), 7.40 (d, 1H, J=8.4 Hz); ³C NMR(CDCl₃) δ 177.1, 173.2, 173.1, 172.7, 172.3, 169.5, 168.9, 101.5, 75.0,74.8, 71.2, 70.9, 69.1, 60.5, 53.1, 51.4,46.1, 41.5, 41.0, 39.2, 34.5,34.3, 34.1, 34.0, 31.9, 31.6, 31.5, 29.8, 29.6, 29.4, 29.0, 28.9, 28.8,25.6, 25.3, 25.1, 25.0, 22.7, 22.6, 14.1, 8.7.

[0257] Anal. Calcd. for C₇₈H₁₄₈N₃O₁₉P: C, 64.04; H, 10.20; N, 2.87; P,2.12. Found: C, 63.77; H, 10.11; N, 2.85; P, 2.02.

EXAMPLE 20 (B19) Preparation of2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0258] (1) 2-Amino-1-(t-butyldiphenylsilyloxy)ethane (330 mg, 1.1 mmol)and (R)-3-tetradecanoyloxytetradecanoic acid (500 mg, 1.11 mmol) weredissolved in CH₂Cl₂ (10 mL) and treated with powdered 4 A molecularsieves (500 mg). After 1 h EEDQ (297 mg, 1.2 mmol) was added and thereaction was stirred for 18 h, filtered through Celite® and concentratedin vacuo. The residue was chromatographed over silica gel using 15%EtOAc/hexanes to give 675 mg (92%) of a colorless solid. A portion ofthis material (500 mg, 0.68 mmol) was deprotected with TBAF (1 M in THF,1 mL, 1 mmol) in THF (5 mL) by stirring at room temperature for 2 h. Thereaction mixture was diluted with Et₂O (50 mL) and washed with brine(2×50 mL). The brine was back extracted with Et₂O (2×50 mL) and thecombined organic extracts were dried over Na₂SO₄ and concentrated invacuo to afford 338 mg (62%) of2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethanol as an off-whitesolid.

[0259] (2) In the same manner as described in Example 2-(6), thecompound prepared in (1) above (338 mg, 0.68 mmol) and the compoundprepared in Example 2-(4) (786 mg, 0.61 mmol) were coupled in thepresence of mercury cyanide (770 mg, 3.05 mmol) to give 245 mg (24%) of2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR(CDCl₃)60.88(t, 12H, J=6.9 Hz), 1.1-1.8(m,84H), 1.81 (s, 3H), 1.89 (s, 3H), 2.15-2.55 (m, 8H), 3.25 (m, 1H), 3.47(m, 2H), 3.67 (m, 1H), 3.83 (m, 2H), 4.28 (dd, 1H, J=12.2, 4.9 Hz), 4.36(d, 1H, J=11.0 Hz), 4.68 (m, 2H), 4.78 (d, 1H, J=11.6 Hz), 4.94 (d, 1H,J=11.6 Hz), 5.16 (m, 2H), 5.53 (t, 1H, J=10.0 Hz), 6.06 (d, 1H, J=4.9Hz), 6.19 (m, 1H), 7.25 (m, 10H).

[0260] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (500 mg, 0.29 mmol) was deprotected withzinc (980 mg, 15 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (155 mg, 0.34 mmol) in thepresence of EEDQ (110 mg, 0.44 mmol) to give 315 mg (62%) of2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-β-D-glucopyranosideas an amorphous solid.

[0261] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (200 mg, 0.113 mmol) was hydrogenated inthe presence of platinum oxide (100 mg) to give 142 mg (76%) of2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-β-D-glucopyranosidetriethylammonium salt as a white solid: mp 175-176° C.; IR (film) 3285,3098, 2955, 2919, 2851, 1731, 1659, 1642, 1556, 1468, 1379, 1250, 1228,1174, 1110, 1083, 1046, 962, 857 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ0.88 (t,18H, J=6.0 Hz), 1.1-1.7(m, 135H),2.2-2.7(m, 15H),3.06(q, 6H, J=7.1Hz),3.2-4.1 (m, 8H), 4.21 (q, 1H, J=9.9 Hz), 4.51 (d, 1H, J=8.2 Hz),5.05-5.25 (m, 4H), 7.33 (d, 1H, J=8.5 Hz), 7.50 (br t, 1H, J=4.8 Hz);¹³C NMR (CDCl₃) δ 173.7, 173.3, 170.6, 170.3, 169.9, 100.9, 75.8, 73.0,71.3, 71.1, 70.9, 70.6, 68.3, 60.6, 55.1, 45.7, 41.6, 41.2, 39.5, 34.6,34.5, 34.4, 32.0, 29.8, 29.4, 29.3, 25.4, 25.1, 22.7, 14.2, 8.6.

[0262] Anal. Calcd. for C₉₈H₁₉₀N₃O₁₇P 2H₂O: C, 67.28; H, 11.18; N, 2.40;P, 1.77. Found: C, 67.01; H, 11.18; N, 2.15; P, 2.01.

EXAMPLE 21 (B20) Preparation of2-[(R)-3-decanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-2-[(R)-3-decanoyloxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0263] (1) In the same manner as described in Example 20-(1),2-amino-1-(t-butyldiphenylsilyloxy)ethane (450 mg, 1.5 mmol) wasacylated with (R)-3-decanoyloxytetradecanoic acid (600 mg, 1.5 mmol) inthe presence of EDC MeI (594 mg, 2.0 mmol) and then deprotected withTBAF (1.0 M in THF, 2.5 mL, 2.5 mmol) in THF (10 mL) to afford 488 mg(81%) of 2-[(R)-3-decanoyloxytetradecanoylamino]ethanol as an off-whitesolid.

[0264] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (385 g, 0.87 mmol) and the compoundprepared in Example 15-(4) (1.05 g, 0.87 mmol) were coupled in thepresence of AgOTf (560 mg, 2.2 mmol) to give 1.04 g (74%) of2-[(R)-3-decanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ0.88 (t, 12H, J=6.9 Hz), 1.1-1.6(m, 68H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.7 Hz), 2.44 (m,2H), 4.34 (m, 5H), 4.72 (m, 2H), 4.83 (q, 1H, J=9.3 Hz), 5.09 (m, 1H),5.51 (t, 1H, J=10.2 Hz),5.79 (d, 1H, J=8.0 Hz),6.26 (d, 1H, J=3.4 Hz),7.31 (m, 10H).

[0265] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (700 mg, 0.44 mmol) was deprotected withzinc (1.42 g, 21.7 mmol) and then acylated with(R)-3-decanoyloxytetradecanoic acid (190 mg, 0.48 mmol) in the presenceof EEDQ (148 mg, 0.6 mmol) to give 432 mg (62%) of2-[(R)-3-decanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-2-[(R)-3-decanoyloxytetradecanoylamino]-β-D-glucopyranosideas an amorphous solid.

[0266] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (400 mg, 0.25 mmol) was hydrogenated inthe presence of platinum oxide (200 mg) to give 200 mg (52%) of2-[(R)-3-decanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-2-[(R)-3-decanoyloxytetradecanoylamino]-β-D-glucopyranosidetriethylammonium salt as a white solid: mp 165-166° C.; IR (film) 3289,3094, 2956, 2922, 2853, 1732, 1658, 1644, 1556, 1467, 1379, 1247, 1164,1107, 1081, 1048 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88(t, 18H, J=6.9 Hz),1.1-1.7 (m, 111H), 2.2-2.7 (m, 15H), 3.05 (q, 6H, J=7.1 Hz), 3.2-3.85(m, 9H), 4.52 (d, 1H, J=8.2 Hz), 5.05-5.25 (m, 4H), 7.21 (d, 1H, J=8.5Hz), 7.42 (br t, 1H);

[0267]¹³C NMR (CDCl₃) δ 173.8, 173.3, 170.7, 170.3, 170.0, 100.9, 75.6,73.0, 71.3, 70.9, 70.6, 68.3, 60.7, 55.0, 45.8, 41.6, 41.2, 39.5, 34.5,34.4, 34.1, 31.9, 29.8, 29.6, 29.5, 29.4, 25.4, 25.1, 22.7, 14.2, 8.6.

[0268] Anal. Calcd. for C₈₆H₁₆₆N₃O₁₇P H₂O: C, 66.08; H, 10.83; N, 2.69;P, 1.98. Found: C, 65.80; H, 10.63; N, 2.63; P, 2.04.

EXAMPLE 22 (B21) Preparation of3-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O, N=1,M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0269] (1) In the same manner as described in Example 20-(1),3-amino-1-(t-butyldiphenylsilyloxy)propane (470 mg, 1.5 mmol) wasacylated with (R)-3-tetradecanoyloxytetradecanoic acid (680 mg, 1.5mmol) in the presence of EDC MeI (595 mg, 2.0 mmol) and then deprotectedwith TBAF (1.0 M in THF, 2.0 mL, 2.0 mmol) in THF (10 mL) to afford 698mg (91%) of 3-[(R)-3-tetradecanoyloxytetradecanoylamino]-1-propanol asan off-white solid.

[0270] (2) In the same manner as described in Example 13-(4), thecompound prepared in Example 2-(3) (7.9 g, 5.88 mmol) was deprotectedwith TFA (10 mL) and then treated with the Vilsmeier reagent generatedfrom DMF (1.8 mL, 23.5 mmol) and oxalyl chloride (1.03 mL, 11.76 mmol)in CH₂Cl₂ (60 mL) to give 6.32 g (85%) of2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosylchloride as a white foam: ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=6.8 Hz),1.2-1.55 (m, 42H), 1.78 (s, 3H), 1.88 (s, 3H), 2.18 (t, 2H, J=7.5 Hz),2.43 (m, 2H), 4.31 (m, 4H), 4.68 (d, 1H, J=11.9 Hz), 4.74 (d, 1H, J=11.9Hz), 4.83 (q, 1H, J=9.3 Hz), 5.09 (m, 1H), 5.51 (t, 1H, J=9.7 Hz), 5.78(d, 1H, J=8.0 Hz), 6.26 (d, 1H, J=3.4 Hz), 7.31 (m, 10H).

[0271] (3) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (613 mg, 1.2 mmol) and the compoundprepared in (2) above (1.5 g, 1.2 mmol) were coupled in the presence ofAgOTf (642 mg, 2.5 mmol) to give 1.43 g (68%) of3-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.9 Hz), 1.1-1.8(m, 86H), 1.82 (s, 3H), 1.89 (s, 3H), 2.20 (t, 2H, J=7.6 Hz), 2.29 (t,2H, J=7.7 Hz), 2.44 (m, 4H), 3.21 (m, 1H), 3.42 (m, 1H), 3.54 (m,2H),3.80(m, 1H), 3.94 (m, 1H), 4.28 (dd, 1H, J=12.3, 5.2 Hz),4.38(d, 1H,J=10.8 Hz), 4.70(m, 3H), 4.81 (d, 1H, J=8.2 Hz),5.14(m, 2H),5.47(t, 1H,J=9.6 Hz),6.13 (d, 1H, J=7.6 Hz), 6.22 (br. s, 1H), 7.25 (m, 10H).

[0272] (4) In the same manner as described in Example 2-(7), thecompound prepared in (3) above (700 mg, 0.40 mmol) was deprotected withzinc (1.32 g, 20.1 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (200 mg, 0.44 mmol) in thepresence of EEDQ (125 mg, 0.5 mmol) to give 435 mg (60%) of3-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosideas an amorphous solid.

[0273] (5) In the same manner as described in Example 2-(8), thecompound prepared in (4) above (400 mg, 0.22 mmol) was hydrogenated inthe presence of platinum oxide (200 mg) to give 170 mg (45%) of3-[(R)-3-tetradecanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosidetriethylammonium salt as a white solid: mp 171-172° C.; IR (film) 3288,3094, 2955, 2919, 2850, 1731, 1658, 1344, 1556, 1468, 1378, 1320, 1251,1226, 1172, 1106, 1083, 1044 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88(t, 18H,J=6.0 Hz), 1.1-1.7(m, 135H),2.2-2.7(m, 15H),3.06(q, 6H, J=7.1 Hz),3.2-4.1 (m, 8H), 4.21 (q, 1H, J=9.9 Hz), 4.51 (d, 1H, J=8.3 Hz),5.05-5.25 (m, 4H), 7.23 (t, 1H, J=5.3 Hz), 7.33 (d, 1H, J=8.6 Hz); ¹³CNMR (CDCl₃) δ 173.5, 173.4, 170.6, 170.2, 169.9, 100.6, 75.8, 71.5,70.9, 70.5, 66.8, 60.4, 55.3, 45.6, 41.4, 39.4, 36.3, 34.6, 34.5, 34.2,31.9, 29.7, 29.4, 29.3, 29.1, 25.4, 25.1, 22.7, 14.1, 8.5.

[0274] Anal. Calcd. for C₉₉H₁₉₂N₃O₁₇P 2H₂O: C, 67.42; H, 11.20; N, 2.38;P, 1.76. Found: C, 66.97; H, 11.01; N, 2.38; P, 1.95.

EXAMPLE 23 (B22) Preparation of4-[(R)-3-tetradecanoyloxytetradecanoylamino]butyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O, N=2,M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0275] (1) In the same manner as described in Example 20-(1),4-amino-1-(t-butyldiphenylsilyloxy)butane (500 mg, 1.53 mmol) wasacylated with (R)-3-tetradecanoyloxytetradecanoic acid (695 mg, 1.53mmol) in the presence of EDC MeI (595 mg, 2.0 mmol) and then deprotectedwith TBAF (1.0 M in THF, 2.5 mL, 2.5 mmol) in THF (15 mL) to afford 651mg (81%) of 4-[(R)-3-tetradecanoyloxytetradecanoylamino]-1-butanol as anoff-white solid.

[0276] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (650 mg, 1.25 mmol) and the compoundprepared in Example 22-(2) (1.6 g, 1.25 mmol) were coupled in thepresence of AgOTf (1.16 g, 4.5 mmol) to give 1.65 g (75%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]butyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.9 Hz), 1.1-1.8(m, 88H), 1.82 (s, 3H),1.89(s, 3H),2.15-2.55(m, 8H),3.24(m, 2H),3.50(m,2H),3.83(m, 2H),4.27(dd, 1H, J=12.1, 3.8 Hz), 4.32 (d, 1H, J=11.5 Hz),4.66 (m, 2H), 4.78 (d, 1H, J=12.1 Hz), 4.89 (d, 1H, J=8.0 Hz), 5.15 (m,2H), 5.54 (t, 1H, J=9.7 Hz), 5.95 (m, 2H), 7.25 (m, 10H).

[0277] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (700 mg, 0.39 mmol) was deprotected withzinc (1.30 g, 19.8 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (195 mg, 0.43 mmol) in thepresence of EEDQ (125 mg, 0.5 mmol) to give 421 mg (60%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]butyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosideas an amorphous solid.

[0278] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (400 mg, 0.22 mmol) was hydrogenated inthe presence of platinum oxide (200 mg) to give 212 mg (55%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]butyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosidetriethylammonium salt as a white solid: mp 171-172° C.; IR (film) 3298,2955, 2920, 2851, 1732, 1645, 1550, 1467, 1378, 1181, 1107, 1083, 1044,721 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88(t, 18H, J=6.9 Hz), 1.1-1.7 (m,135H), 2.2-2.7 (m, 19H), 3.05 (q, 6H, J=7.1 Hz), 3.18 (m, 2H), 3.3-3.5(m, 6H), 3.78 (m, 3H), 3.97 (d, 1H, J=12.5 Hz), 4.23 (q, 1H, J=10.0 Hz),4.50 (d, 1H, J=8.5 Hz), 5.13 (m, 4H), 7.12 (d, 1H, J=9.1 Hz); ¹³C NMR(CDCl₃) δ 173.9, 173.4, 173.3, 170.8, 169.9, 169.8, 101.0, 75.6, 73.2,71.4, 71.1, 70.6, 68.9, 60.7, 54.8, 45.9, 41.5, 39.6, 38.9, 34.6, 34.3,32.0, 29.8, 29.5, 29.0, 28.9, 26.3, 25.4, 25.1, 22.7, 14.2, 8.7.

[0279] Anal. Calcd. for C₁₀₀H₁₉₄N₃O₁₇P H₂O: C, 68.26; H, 11.23; N, 2.39;P, 1.76. Found: C, 68.21; H, 11.03; N, 2.26; P, 1.73.

EXAMPLE 24 (B23) Preparation of4-[(R)-3-tetradecanoyloxytetradecanoylamino]hexyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O, N=4,M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0280] (1) In the same manner as described in Example 20-(1),6-amino-1-(t-butyldiphenylsilyloxy)hexane (1.48 g, 4.15 mmol) wasacylated with (R)-3-tetradecanoyloxytetradecanoic acid (2.07 g, 4.56mmol) in the presence of EDC MeI (1.35 g, 4.56 mmol) and thendeprotected with TBAF (1.0 M in THF, 1.53 mL, 1.53 mmol) in THF (46 mL)to afford 700 mg (30%) of6-[(R)-3-tetradecanoyloxytetradecanoylamino]-1-hexanol as an off-whitesolid.

[0281] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (689 mg, 1.20 mmol) and the compoundprepared in Example 22-(2) (1.25 g, 1.00 mmol) were coupled in thepresence of AgOTf (1.28 g, 5.0 mmol) to give 1.59 g (94%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]hexyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.6 Hz), 1.1-1.8(m, 92H), 1.82 (s, 3H), 1.89 (s, 3H), 2.22 (t, 2H, J=7.6 Hz), 2.29 (t,2H, J=7.4 Hz), 2.45 (m, 4H), 3.22 (m, 1H),3.46(m, 2H),3.83 (m,2H),3.94(m, 1H), 4.31 (m, 2H),4.64(m, 2H),4.83 (d, 1H, J=12.1 Hz), 4.97(d, 1H, J=7.8 Hz), 5.17 (m, 2H), 5.59 (t, 1H, J=8.8 Hz), 5.75 (m, 1H),5.84 (d, 1H, J=7.6 Hz), 7.25 (m, 10H).

[0282] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (1.57 g, 0.88 mmol) was deprotected withzinc (2.88 g, 44.1 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (481 mg, 1.06 mmol) in thepresence of EEDQ (327 mg, 1.32 mmol) to give 1.57 g (97%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]hexyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino])-β-D-glucopyranosideas an amorphous solid.

[0283] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (1.57 g, 0.85 mmol) was hydrogenated inthe presence of platinum oxide (157 mg) to give 130 mg (10%) of4-[(R)-3-tetradecanoyloxytetradecanoylamino]hexyl2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranosidetriethylammonium salt as a white solid: mp 150-152° C.; IR(film) 3284,3099, 2954, 2920, 2851, 1731, 1657, 1637, 1557, 1467, 1418, 1378, 1320,1249, 1179, 1108, 1083, 1044, 856, 721 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.89(t, 18H, J=6.6 Hz), 1.1-1.7 (m, 135H), 2.2-2.7 (m, 23H), 3.05 (q, 6H,J=7.1 Hz), 3.18 (m, 2H), 3.39 (d, 1H, J=8.2 Hz), 3.49 (q, 1H, J=7.5 Hz),3.82 (m, 2H), 3.99 (d, 1H, J=11.9 Hz), 4.25 (q, 1H, J=8.9 Hz), 4.59 (m,2H), 5.18 (m, 4H); ¹³C NMR (CDCl₃) δ 173.7, 173.3, 170.6, 169.7, 169.4,100.6, 75.5, 73.1, 71.3, 70.9, 70.6, 69.2, 60.6, 55.2, 45.8, 41.7, 41.4,39.5, 39.4, 34.6, 34.3, 34.2, 34.1, 31.9, 29.7, 29.4, 29.2, 26.5, 25.5,25.3, 25.1, 22.7, 14.1,8.6.

[0284] Anal. Calcd. for C₁₀₂H₁₉₈N₃O₁₇P H₂O: C, 68.53; H, 11.28; N, 2.33;P, 1.73. Found: C, 68.63; H, 11.12; N, 2.26; P, 1.66.

EXAMPLE 25 (B24) Preparation ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]β-D-glucopyranosyl]-L-serinamideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CONH₂, R₈=PO₃H₂).

[0285] (1) A suspension of L-serinamide hydrochloride (0.157 g, 1.18mmol) and (R)-3-tetradecanoyloxytetradecanoic acid (0.61 g, 1.34 mmol)in CH₂Cl₂ (6 mL) was treated with triethylamine (0.18 mL, 1.3 mmol) andthe resulting solution was stirred with 4 Å molecular sieves for 30 min.EEDQ (0.437 g, 1.77 mmol) was then added and the mixture was stirred for16 h at room temperature. The product that precipitated was collectedand washed with CH₂Cl₂ (2×25 mL) to give 0.455 g (71%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-L-serinamide as a colorlesspowder: mp 126-130° C.; ¹H NMR (CDCl₃) δ 0.88 (t, 6H, J=7 Hz), 1.15-1.7(m, 42H), 2.31 (t, 2H, J=7.5 Hz), 2.51 (d, 2H, J=6.3 Hz), 3.56 (br s,1H), 3.65 (dd, 1H, J=11.2, 5.5 Hz), 3.86 (dd, 1H, J=11.2, 4.5 Hz), 4.21(s, 2H), 4.40 (m, 1H), 5.22 (m, 1H).

[0286] (2) In the same manner as described in Example 2-(6), thecompound prepared in (1) above (0.23 g, 0.246 mmol) and the compoundprepared in Example 2-(4) (0.961 g, 0.745 mmol) were coupled in thepresence of mercury cyanide (0.43 g, 1.7 mmol) to give 0.527 g (71%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2,-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinamideas an amorphous solid: ¹H NMR (CDCl₃) δ0.88 (t, 12H, J=7H),1.0-1.7 (m,84H), 1.80 and 1.89 (2s, 6H), 2.21 (t, 2H, J=7.5 Hz), 2.30 (t, 2H, J=7.5Hz), 2.37 (m, 2H), 2.47 (m, 2H), 3.54 (m, 1H), 3.68 (dd, 1H, J=8, J=11Hz), 3.86 (br d, 1H, J=11 Hz), 4.16 (dd, 1H, J=11, 4 Hz), 4.24 (dd, 1H,J=12, 4.3 Hz), 4.40 (d, 1H, J=12 Hz), 4.6-4.8 (m, 4H), 5.00 (d, 1H, J=8Hz), 5.1-5.25 (m, 2H), 5.4-5.55 (m, 2H), 5.84 (br s, 1H), 6.61 (br s,2H), 7.1-7.35 (m, 10H).

[0287] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (0.44 g, 0.254 mmol) was deprotected withzinc (0.83 g, 13 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (0.14 g, 0.31 mmol) in thepresence of EEDQ (0.095 g, 0.38 mmol) to give 0.271 g (59%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinamideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 18H, J=6.5 Hz), 1.0-1.7(m, 126H), 2.03 (br s, 1H), 2.15-2.55 (m, 12H), 3.5-4.05 (m, 5H), 4.14(dd, 1H, J=10, 3.5 Hz), 4.5-4.65 (m, 2H), 4.68 (d, 1H, J=8.1 Hz),5.05-5.25 (m, 3H), 5.31 (t, 1H, J=10 Hz), 5.58 (br s, 1H), 6.31 (d, 1H,J=8 Hz), 6.85-6.95 (m, 2H), 7.1-7.4 (m, 10H).

[0288] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (0.25 g, 0.14 mmol) was hydrogenated inthe presence of platinum oxide (0.125 g) to give 0.195 (80%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinamidetriethylammonium salt as a colorless solid: mp 190-191° C. (dec); IR(film) 3418, 3293, 2921, 2850, 1732, 1717, 1651, 1636, 1557, 1540, 1458,1165, 1033 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=7 Hz), 1.0-1.7(m, 135H), 2.2-2.7 (m, 12H), 3.05 (q, 6H, J=7.2 Hz), 3.2-3.45 (m),3.5-4.15 (m, 5H), 4.21 (q, 1H, J=-10 Hz), 4.53 (d, 1H, J=8.1 Hz), 4.58(m, 1H), 5.0-5.3 (m, 4H), 7.25 (d, 1H, J=8.4 Hz), 7.40 (d, 1H, J=7.2Hz); ³C NMR (CDCl₃—CD₃OD) δ 173.7,173.5,172.5, 170.7, 170.5, 170.4,101.4, 75.5, 73.4, 71.1, 70.9, 70.2, 68.6, 60.0, 53.9, 52.2, 45.6, 41.2,41.0, 38.9, 34.4, 34.2, 31.8, 29.6, 29.5, 29.3, 29.1, 25.2, 24.9, 22.6,14.0, 8.3.

[0289] Anal. Calcd for C₉₉H₁₉₁N₄O₁₈P·2.5H₂O: C, 66.00; H, 10.97; N,3.11; P, 1.72. Found: C, 66.04; H, 10.99; N, 3.03; P, 1.95.

EXAMPLE 26 (B25) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinamideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CONH₂, R₈=PO₃H₂).

[0290] (1) In the same manner as described in Example 25-(1),L-serinamide hydrochloride (169 mg, 1.2 mmol) was acylated with(R)-3-decanoyloxytetradecanoic acid (478 mg, 1.2 mmol) in the presenceof EEDQ (371 mg, 1.5 mmol) in CH₂Cl₂ to afford 428 mg (74%) ofN-[(R)-3-decanoyloxytetradecanoyl]-L-serinamide as a white solid: ¹H NMR(CDCl₃) δ 0.88 (t, 6H), 1.1-1.7 (m, 34H), 2.33 (t, 2H, J=7.5 Hz), 2.54(d, 2H, J=6.6 Hz), 3.35 (s, 2H), 3.72 (dd, 1H, J=11.0, 5.2 Hz), 3.84(dd, 1H, J=11.3, 5.0 Hz), 4.20 (t, 1H, J=5.1 Hz), 5.26 (t, 1H, J=6.4Hz).

[0291] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (410 mg, 0.85 mmol) and the compoundprepared in Example 15-(4) (1.05 g, 0.87 mmol) were coupled in thepresence of AgOTf (560 mg, 2.2 mmol) to afford 780 g (56%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinamideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H), 1.1-1.6 (m, 68H),1.80 (s, 3H), 1.89 (s, 3H), 2.30 (m, 8H), 3.53 (m, 1H), 3.68 (m, 1H),3.85 (br. d, 1H, J=9.4 Hz), 4.15 (dd, 1H, J=10.8, 3.7 Hz), 4.24 (dd, 1H,J=12.3, 4.6 Hz), 4.40 (d, 1H, J=10.8), 4.65 (m, 4H), 5.00 (d, 1H, J=8.2Hz), 5.18 (m, 2H), 5.46 (m, 2H), 5.83 (m, 1H), 6.60 (m, 2H), 7.30 (m,10H).

[0292] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (600 mg, 0.36 mmol) was deprotected withzinc (1.19 g, 18.2 mmol) and acylated with(R)-3-decanoyloxytetradecanoic acid (160 mg, 0.4 mmol) in the presenceof EEDQ (124 mg, 0.50 mmol) to afford 371 mg (62%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinamideas an amorphous solid.

[0293] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (330 mg, 0.20 mmol) was hydrogenated inthe presence of platinum oxide (200 mg) to afford 120 mg (44%) ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinamidetriethylammonium salt as a white powder: mp 187-189° C.; IR (film) 3419,3286, 3220, 3098, 2955, 2922, 2852, 1732, 1680, 1662, 1644, 1559, 1467,1247, 1167, 1107, 1080, 1051, 965, 913 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ0.89(t, 18H, J=7.0 Hz), 1.1-1.7(m, 111H), 2.2-2.7 (m, 12H),3.07(q, 6H,J=7.1 Hz), 3.68 (m, 1H), 3.87 (m, 1H), 4.09 (dd, 1H, J=10.8, 3.6 Hz),4.22 (m, 1H), 4.53 (d, 1H, J=8.2 Hz), 4.58 (m, 1H), 5.13 (m, 3H), 5.28(m, 1H), 7.53 (d, 1H, J=9.0 Hz), 7.56 (d, 1H, J=7.7 Hz); ¹³C NMR(CDCl₃)δ 173.5, 173.2, 170.2, 169.8, 102.3, 75.7, 73.5, 71.3, 70.7, 70.1, 68.8,60.8, 53.9, 51.7, 45.8, 41.5, 41.1, 39.1, 34.6, 34.5, 34.2, 32.0, 29.7,29.6, 29.5, 29.4, 25.7, 25.4, 25.1, 22.7, 14.1, 8.6.

[0294] Anal. Calcd. for C₈₇H₁₆₇N₄O₁₈P H₂O: C, 65.05; H, 10.60; N, 3.49;P, 1.93. Found: C, 65.06; H, 10.40; N, 3.31; P, 2.00.

EXAMPLE 27 (B26) Preparation ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-1-serineMethyl Ester Triethylammoniun Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂ME, R₈=PO₃H₂).

[0295] (1) A solution of the compound prepared in Example 12-(2) (0.290g, 0.157 mmol) in THF (20 mL) was hydrogenated in the presence of 5%palladium on carbon (50 mg) at room temperature and atmospheric pressurefor 3 h. The catalyst was removed by filtration and the filtrateconcentrated. A solution of the residue in CHCl₃ (5 mL) at 0° C. wastreated with a solution of diazomethane (0.5 mmol) in ether (5 mL) andthen stirred for 30 min at 0° C. AcOH (0.5 mL) was added and theresulting colorless solution was diluted with ether (50 mL), washed withsaturated aqueous NaHCO₃ (25 mL), dried (Na₂SO₄) and concentrated. Flashchromatography on silica gel (gradient elution, 20→25% EtOAcs-hexanes)afforded 0.199 g (72%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichoro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosyl]-L-serinemethyl ester as an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=6.5Hz), 1.1-1.75 (m, 84H), 1.81 and 1.89 (2s, 6H), 2.36 (t, 2H, J=7.5 Hz),2.25-2.6 (m, 6H), 3.48 (q, 1H, J=8 Hz), 3.7-3.9 (m, 5H), 4.2-4.4 (m,3H), 4.6-4.85 (m, 4H), 4.88 (d, 1H, J=7.8 Hz), 5.03-5.22 (m, 2H), 5.49(t, 1H, J=9.5 Hz), 6.21 (br s, 1H), 6.59 (d, 1H, J=7.8 Hz), 7.1-7.4 (m,10H).

[0296] (2) In the same manner as described in Example 2-(7), thecompound prepared in (1) above (0.195 g, 0.111 mmol) was deprotectedwith zinc (0.36 g, 5.5 mmol) and acylated with(R)-3-tetradecanoyloxytetradecanoic acid (0.060 g, 0.13 mmol) in thepresence of EEDQ (0.041 g, 0.17 mmol) to give 0.138 g (69%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[(R)-4-O-diphenylphosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl-β-D-glucopyranosyl]-L-serinemethyl ester as an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 18H, J=6.5Hz), 1.0-1.75 (m, 126H), 2.15-2.45 (m, 10H), 2.52 (dd, 1H, J=14.7, 6Hz), 2.66 (dd, 1H, J=14.7, 6 Hz), 3.35 (br s, 1H), 3.4-3.8 (m, 7H), 3.88(dd, 1H, J=11 Hz), 4.18 (dd, 1H, J=1 Hz), 4.6-4.75 (m, 2H), 5.03 (d, 1H,J=7.8 Hz), 5.1-5.25 (m, 3H), 5.50 (t, 1H, J=˜9.5 Hz), 6.50 (d, 1H, J=7.2Hz), 6.97 (d, 1H, J=7.8 Hz), 7.1-7.4 (m, 10H).

[0297] (3) In the same manner as described in Example 2-(8), thecompound prepared in (2) above (0.100 g, 0.055 mmol) was hydrogenated inthe presence of platinum oxide (50 mg) to give 0.055 g (57%) ofN-[(R)-3-tetradecanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosyl]-L-serinemethyl ester triethylammonium salt as a colorless solid: mp 142-143° C.(dec); IR (film) 3289, 2955, 2921, 2852, 1733, 1718, 1699, 1652, 1558,1540, 1521, 1506, 1469, 1457, 1375, 1360, 1259 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.5 Hz), 1.0-1.7 (m, 135H), 2.2-2.7 (m,12H), 3.05 (q, 6H, J=7.5 Hz), 3.31 (d, 1H, J=9.3 Hz), 3.37 (s, 1H),3.55-3.9 (m, 10H), 3.97 (d, 1H, J=12 Hz), 4.1-4.25 (m, 2H), 4.55-4.65(m, 2H), 5.05-5.25 (m, 3H), 7.23 (d, 1H, J=8.1 Hz), 7.47 (d, 1H, J=7.2Hz); ¹³C NMR (CDCl₃) δ 173.6, 173.4, 170.5, 170.4, 170.1, 100.7, 75.9,72.8, 71.2, 70.8, 70.6, 68.5, 60.3, 55.3, 52.7, 52.4, 47.7, 41.5, 40.9,39.7, 34.6, 34.5, 34.3, 32.0, 29.8, 29.4, 25.4, 25.1, 22.7, 14.2, 8.5.

[0298] Anal. Calcd for C₁₀₀H₁₉₂N₃O₁₉P·H₂O: C, 67.11; H, 10.93; N, 2.35;P, 1.73. Found: C, 66.91; H, 10.93; N, 2.31; P, 2.11.

EXAMPLE 28 (B27) Preparation ofN-(carboxymethyl)-N-[(R)-3-tetradecanoyloxytetradecanoyl]-2-aminoethyl2-deoxy-4-O-phophono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-d-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₁₃H₂₇CO, X=Y=O,N=M=P=0, R₄=R₅=R₆=R₉=H, R₇=CO₂H, Q=1, R₈=PO₃H₂).

[0299] (1) In the same manner as described in Example 2-(5),N-(2-hydroxyethyl)glycine t-butyl ester (0.25 g, 1.43 mmol) was acylatedwith (R)-3-tetradecanoyloxytetradecanoic acid (0.714 g, 1.57 mmol) inthe presence of EDC MeI (0.466 g, 1.57 mmol) to give 0.46 g (51%) ofN-(2-hydroxyethyl)-N-[(R)-3-tetradecanoyloxytetradecanoyl]glycinet-butyl ester as an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 6H,J=˜6.5 Hz), 1.15-1.7 (m, 51H), 2.26 (t, 2H, J=7.5 Hz), 2.60 (dd, 1H, J6.5, 15 Hz), 2.86 (dd, 1H, J=6.7, 15 Hz), 3.40-4.15 (m, 7H), 5.25 (m,1H).

[0300] (2) In the same manner as described in 13-(5), the compoundprepared in (1) above (0.21 g, 0.334 mmol) and the compound prepared inExample 22-(2) (0.458 g, 0.368 mmol) were coupled in the presence ofAgOTf (0.688 g, 2.68 mmol) to give 0.39 g (64%) ofN-(t-butyloxycarbonylmethyl)-N-[(R)-3-tetradecanoyloxytetradecanoyl]-2-aminoethyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosideas an amorphous solid: ¹H NMR (CDCl₃) δ 0.88 (t, 12H, J=˜6.5 Hz),1.0-1.95 (m, 99H), 2.1-2.6 (m, 7H), 2.84 (dd, 1H, J=5, 15 Hz), 3.2-4.15(m, 8H), 4.15-4.45 (m, 2H), 4.55-4.9 (m, 3H), 5.00 (d, 1H, J=8 Hz), 5.13(m, 2H), 5.4-5.65 (m, 1H), 6.16 (d, 1H, J=7 Hz), 7.05-7.4 (m, 10H).

[0301] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (0.339 g, 0.185 mmol) was deprotectedwith zinc (0.36 g, 5.54 mmol) and then acylated with(R)-3-tetradecanoyloxytetradecanoic acid (0.100 g, 0.221 mmol) in thepresence of EEDQ (0.068 g, 0.276 mmol) to give 0.25 g (71%) ofN-(t-butyloxycarbonylmethyl)-N-[(R)-3-tetradecanoyloxytetradecanoyl]-2-aminoethyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosideas a colorless solid.

[0302] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (0.25 g, 0.131 mmol) was hydrogenated inthe presence of platinum oxide (125 mg) in 9:1 THF-AcOH (15 mL). Thecrude hydrogenolysis product was dissolved in CH₂Cl₂ (1 mL), cooled to0° C., and treated dropwise with TFA (0.5 mL). After stirring for 2 h at0° C., the reaction mixture was concentrated and residual TFA wasremoved by azeotroping with toluene. The resulting residue (0.23 g) wasdissolved in 1% aqueous triethylamine (12 mL) and lyophilized. Flashchromatography on silica gel withchloroform-methanol-water-triethylamine (91:8:0.5:0.5→85:15:0.5:0.5,gradient elution) and further purification by means of acidic extractionas described in Example 2-(8) and lyophilization from 1% aqueoustriethylamine (6 mL) afforded 99 mg (43%) ofN-(carboxymethyl)-N-[(R)-3-tetradecanoyloxytetradecanoyl]-2-aminoethyl2-deoxy-4-O-phosphono-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as colorless solid: mp 162-163° C. (dec); IR(film) 3286, 2922, 2852, 1732, 1651, 1556, 1455, 1434, 1378, 1260, 1088,801 cm⁻¹; ¹H NMR (CDCl₃) δ 0.88 (t, 18H, J=6.5 Hz), 1.0-1.75 (m, 135H),2.2-3.0 (m, 14H), 3.04 (q, 6H, J=7.2 Hz), 3.25-3.8 (m, 5H), 3.85-4.3 (m,5H), 4.55 (d, 1H, J=7.5 Hz), 4.68 (d, 1H, J=8.1 Hz), 5.05-5.35 (m, 4H).

[0303] Anal. Calcd for C₁₀₀H₁₉₂N₃O₁₉P·3H₂O: C, 65.79; H, 10.60; N, 2.30;P, 1.70. Found: C, 65.82; H, 10.44; N, 2.40; P, 1.79.

EXAMPLE 29 (B28) Preparation ofN-Carboxymethyl-N-[(R)-3-decanoyloxytetradecanoyl]-3-aminopropyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino])-3-O-[(R)-3-decanoyoxytetradecanoyl]-β-d-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O, N=1,M=P=0, R₄=R₅=R₆=R₉=H, R₇=CO₂H, Q=1, R₈=PO₃H₂).

[0304] (1) In the same manner as described in Example 2-(5),N-(3-hydroxypropyl)glycine benzyl ester (450 mg, 2.0 mmol) was acylatedwith (R)-3-decanoyloxytetradecanoic acid (1.0 g, 2.5 mmol) in thepresence of EDC MeI (900 mg, 3.0 mmol) in CH₂Cl₂ to afford 0.76 g (63%)of N-(3-hydroxypropyl)-N-[(R)-3-decanoyloxytetradecanoyl]glycine benzylester as a colorless oil: ¹H NMR (CDCl₃) (1:1 mixture of rotomers) δ0.88 (t, 6H, J=6.6 Hz), 1.1-1.7 (m, 35H), 1.78 (m, 1H), 2.26 (q, 2H,J=7.6 Hz), 2.37 and 2.54 (2 dd, 1H, J=14.9, 6.9 Hz), 2.60 and 2.89 (2dd, 1H, J=14.8, 6.0 Hz), 3.51 (m, 4H), 3.70 (m, 1H), 3.95-4.25 (m, 2H),5.1-5.25 (m, 3H), 7.35 (m, 5H).

[0305] (2) In the same manner as described in Example 13-(5), thecompound prepared in (1) above (500 mg, 0.83 mmol), and the compoundprepared in Example 15-(4) (1.0 g, 0.83 mmol) were coupled in thepresence of AgOTf(1.07 g, 4.15 mmol) to afford 1.27 g (72%) ofN-(benzyloxycarbonylmethyl)-N-[(R)-3-decanoyloxytetradecanoyl]-3-aminopropyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-β-D-glucopyranosidebenzyl ester: ¹H NMR (CDCl₃) (2:1 mixture of rotomers) δ 0.88 (t, 12H,J=6.9 Hz), 1.1-1.7(m, 69H), 1.80(s, 3H), 1.88(s, 3H),2.1-2.6(m,11H),2.81 (dd, 1H, J=14.8, 6.2 Hz), 3.37 (m, 1H), 3.52 (m, 2H), 3.76 (m,1H), 3.87 (m, 1H), 4.05 (m, 2H), 4.28 (m, 3H), 4.62 (m, 3H), 4.77 (m,1H), 4.93 (d, 1H, J=8.2 Hz), 5.15 (m, 4H), 5.46 and 5.61 (2 t, 1H, J=9.5Hz), 5.95 and 6.05 (2 d, 1H, J=7.5 Hz), 7.1-7.4 (m, 15H).

[0306] (3) In the same manner as described in Example 2-(7), thecompound prepared in (2) above (1.25 g, 0.71 mmol) was deprotected withzinc (2.31 g, 3.53 mmol) and acylated with(R)-3-decanoyloxytetradecanoic acid (353 mg, 0.89 mmol) in the presenceof EEDQ (264 mg, 1.07 mmol) to afford 670 mg (54%) ofN-benzyloxycarbonylmethyl-N-[(R)-3-decanoyloxytetradecanoyl]-3-aminopropyl2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyoxytetradecanoyl]-2-[(R)-3-decanoyloxytetradecanoylamino])-β-D-glucopyranosideas an amorphous solid.

[0307] (4) In the same manner as described in Example 2-(8), thecompound prepared in (3) above (670 mg, 0.38 mmol) was hydrogenated inthe presence of palladium hydroxide on carbon (270 mg) and platinumoxide (200 mg) in EtOH/AcOH (10:1) to afford 240 mg (39%) ofN-carboxymethyl-N-[(R)-3-decanoyloxytetradecanoyl]-3-aminopropyl2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino])-3-O-[(R)-3-decanoyoxytetradecanoyl]-β-D-glucopyranosidetriethylammonium salt as a white powder: mp 156-157° C.; IR (film) 3284,2929, 2853, 2729, 1732, 1655, 1628, 1551, 1466, 1378, 1314, 1164, 1108,1047, 955, 844, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.9Hz), 1.1-1.7(m, 111H), 2.27 (q, 6H, J=6.2 Hz),2.35-2.80 (m, 9H), 3.05(q, 6H, J=7.2 Hz), 3.25-3.60 (m, 4H), 3.75-4.10 (m, 4H), 4.23 (m, 2H),4.47 (d, 1H, J=8.2 Hz), 4.61 (d, 1H, J=8.3 Hz), 5.05-5.25 (m, 4H); ¹³CNMR (CDCl₃) δ 173.4, 173.0, 171.1, 170.6, 170.3, 169.6, 100.5, 74.5,73.9, 71.4, 71.2, 70.7, 70.2, 67.0, 65.8, 60.7, 54.6, 54.3, 51.4, 49.2,46.0, 45.4, 42.1, 41.2, 39.4, 38.0, 37.7, 34.5, 34.3, 34.2, 31.9, 29.8,29.7, 29.6, 29.5, 29.2, 28.1, 25.4, 25.3, 25.1, 22.7, 14.1, 11.1, 8.6.

[0308] Anal. Calcd. for C₈₉H₁₇₀N₃O₁₉P H₂O: C, 65.37; H, 10.60; N, 2.57;P, 1.89. Found: C, 65.35; H, 10.42; N, 2.43; P, 2.05.

EXAMPLE 30 (B29) Preparation of N-[(R)-3hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinamideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₅H₁₁CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CONH₂, R₈=PO₃H₂).

[0309] In the same manner as described in Example 26 and cognate steps,N-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinamidetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serinamide hydrochloride, and (R)-3-hexanoyloxytetradecanoic acid: mp184-185° C.; IR (film) 3416, 3284, 3210, 3096, 2954, 2923, 2853, 1735,1721, 1680, 1664, 1646, 1560, 1466, 1246, 1169, 1080, 1038 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.90(m, 18H), 1.1-1.7 (mH), 2.23-2.47 (m, 6H), 2.48-2.7(m, 6H), 3.06 (q, 6H, J=6 Hz), 3.26-3.34 (mH), 3.66 (m, 1H), 3.77 (d,1H, J=9.5 Hz), 3.82-3.96 (m, 2H), 4.12 (dd, 1H, J=2, 8 Hz), 4.21 (q, 1H,J=8 Hz), 4.56 (d, 1H, J=7 Hz), 4.61 (m, 1H), 5.05-5.18 (m, 3H), 5.24 (m,1H), 7.26 (d, 1H, J=6.5 Hz), 7.40 (d, 1H, J=5.7 Hz).

[0310] Anal. Calcd. for C₇₅H₁₄₃N₄O₁₈P H₂O: C, 62.65; H, 10.16; N, 3,90;P, 2.15. Found: C, 62.60; H, 9.97; N, 3.72; P, 2.25.

EXAMPLE 31 (B30) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinamideTriethylammonium Salt (Compound (I), R₁=R₃=N—C₉H₁₉CO R₂=N—C₅H₁₁CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CONH₂, R₈=PO₃H₂).

[0311] In the same manner as described in Example 26 and cognate steps,N-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinamidetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serinamide hydrochloride, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 200-201° C. dec; IR (film) 3420, 3286, 2956, 2923, 2853, 1733,1680, 1663, 1645, 1556, 1466, 1418, 1378, 1248, 1168, 1106, 1081, 1051,859, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (t, 18H, J=6.9 Hz), 1.0-1.7(m, 103H), 2.15-2.71 (m, 12H), 3.06 (q, 6H, J=7 Hz), 3.68 (m, 1H), 3.87(m, 1H), 4.09 (dd, 1H, J=10.8, 3.6 Hz), 3.35-4.0 (mH), 4.15-4.3 (m, 2H),4.57-4.7 (m, 2H), 5.05-5.3 (m, 4H), 7.42 (m, 1H); ¹³C NMR (CDCl₃) δ173.5, 173.1, 170.2, 169.8, 102.2, 75.8, 73.7, 71.3, 70.7, 70.2, 69.0,60.7, 53.9, 51.7, 45.8, 41.3, 41.1, 39.1, 34.6, 34.5, 34.2, 32.0, 29.7,32.0, 31.4, 29.8, 29.6, 29.5, 29.4, 25.6, 25.4, 25.1, 24.7, 22.7, 22.4,13.9, 8.6.

[0312] Anal. Calcd. for C₈₃H₁₅₉N₄O₁₈P·H₂O: C, 64.31; H, 10.47; N, 3.61.Found: C, 64.31; H, 10.27; N, 3.41.

EXAMPLE 32 (B31) Preparation of2-[(R)-3-hexanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-[(R)-3-hexanoyloxytetradecanoylamino]-α-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₅H₁₁CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0313] In the same manner as described in Example 21 and cognate steps,2-[(R)-3-hexanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-3-O-[(R)-3-hexanoyloxytetradecanoyl]-2-[(R)-3-hexanoyloxytetradecanoylamino]-α-D-glucopyranosidetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino-1-(t-butyldiphenylsilyloxy)ethane,and (R)-3-hexanoyloxytetradecanoic acid: mp 161-162° C.; IR (film) 3288,3096, 2956, 2924, 2854, 1732, 1657, 1645, 1557, 1466, 1378, 1316, 1245,1173, 1080, 1041 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.89 (m, 18H), 1.15-1.67(m, 87H), 2.23-2.70 (m, 15H), 3.06 (q, 6H, J=7.4 Hz), 3.2-3.85 (m, 9H),4.52 (d, 1H, J=8.0 Hz), 5.05-5.27 (m, 4H), 7.24 (d, 1H, J=8.5 Hz), 7.43(br t, 1H); ¹³C NMR (CDCl₃) δ 173.7, 173.3, 173.3, 170.6, 170.2, 169.9,100.9, 75.6, 73.0, 71.3, 70.9, 70.6, 68.3, 60.7, 55.0, 45.8, 41.6, 41.2,39.5, 34.5, 34.4, 34.4, 31.9, 31.3, 29.7, 29.4, 25.4, 24.7, 22.7, 22.4,14.1, 8.6.

[0314] Anal. Calcd. for C₇₄H₁₄₂N₃O₁₇P·H₂O: C, 63.72; H, 10.40; N, 3.01;P, 2.22. Found: C, 63.72; H, 10.21; N, 2.96; P, 2.46.

EXAMPLE 33 (B32) Preparation of2-[(R)-3-hexadecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-2-[(R)-3-octadecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-═-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=C₁₃H₂₇CO, R₂=C₁₇H₃₅CO,R₃=N—C₁₅H₃₁CO, X=Y=O, N=M=P=Q=0, R₄=R₅=R₆=R₇=R₉=H, R₈=PO₃H₂).

[0315] In the same manner as described in Example 21 and cognate steps,2-[(R)-3-hexadecanoyloxytetradecanoylamino]ethyl2-deoxy-4-O-phosphono-2-[(R)-3-octadecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-α-D-glucopyranosidetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,2-amino-1-(t-butyldiphenylsilyloxy)ethane, and (R)-3-tetra-, octa- andhexadecanoyloxytetradecanoic acids: mp 180-181° C.; IR (film) 3284,3097, 2920, 2851, 1731, 1657, 1699, 1683, 1653, 1558, 1541, 1521, 1506,1467, 1435, 1418, 1376, 1258, 1173, 1033 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ0.8-1.75 (mH), 2.2-2.7 (mH), 3.08 (q, 6H, J=7.2 Hz), 3.2-3.5 (m, 5H),3.55-4.05 (mH), 4.24 (q, 1H, J=7 Hz), 4.53 (d, 1H, J=8 Hz), 5.05-5.3 (m,4H), 7.32 (d, 1H, J=9 Hz), 7.49 (br t, 1H); ¹³C NMR (CDCl₃) δ 173.8,173.4, 173.3, 170.6, 170.3, 169.9, 100.9, 75.7, 73.0, 71.3, 70.9, 70.6,68.3, 60.7, 55.0, 45.8, 41.3, 39.5,34.6, 34.4, 32.0, 29.8, 29.4, 25.4,25.1, 22.7, 14.2, 8.6.

[0316] Anal. Calcd. for C₁₀₄H₂₀₂N₃O₁₇P·4H₂O: C, 66.81; H, 11.32; N,2.25. Found: C, 66.52; H, 10.80; N, 2.19.

EXAMPLE 34 (B34) Preparation ofN-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₂=R₃=N—C₅H₁₁CO, X=Y=O,N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂)

[0317] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexanoyloxytetradecanoic acid: mp159-160° C.; IR (film) 3317, 2954, 2924, 2854, 1734, 1654, 1540, 1466,1377, 1316, 1245, 1173, 1082, 846, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88(m, 18H), 1.15-1.7 (mH), 2.2-2.75 (m, 12H), 3.08 (q, 6H, J=7.2 Hz), 3.40(d, 1H, J=9.9 Hz), 3.55-3.95 (mH), 4.15-4.3 (m, 1H), 4.51 (d, 1H, J=8.0Hz), 4.63 (br. s, 1H), 5.1-5.3 (m, 4H), 7.01 (d, 1H, J=9.1 Hz), 7.37 (d,1H, J=8.8 Hz); ¹³C NMR (CDCl₃) δ 177.0, 173.2, 173.2, 172.7, 172.3,169.6, 169.0, 101.5, 75.0, 71.2, 70.9, 70.8, 69.1, 60.5, 53.1, 51.4,46.1, 41.4, 41.0, 39.1, 34.5, 34.2, 34.1, 34.0, 31.9, 31.4, 31.3, 29.8,29.6, 29.4, 25.6, 25.3, 25.1, 24.7, 24.7, 22.7, 22.5, 22.4, 14.1, 14.0,8.7.

[0318] Anal. Calcd. for C₇₅H₁₄₂N₃O₁₉P·2H₂O: C, 61.83; H, 10.10; N, 2.88;P, 2.13. Found: C, 62.07; H, 10.01; N, 2.94; P, 2.40.

EXAMPLE 35 (B35) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=N—C₅H₁₁CO, R₂=R₃=N—C₉H₁₉CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0319] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 158-159° C.; IR (film) 3304, 2956, 2923, 2853, 1732, 1658,1547, 1466, 1378, 1317, 1246, 1174, 1082, 960, 846, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.15-1.7 (mH), 2.2-2.75 (m, 12H), 3.06(q, 6H, J=7.2 Hz), 3.3-3.63 (mH), 3.66-3.98 (m, 4H), 4.1-4.3 (m, 2H),4.54 (d, 1H, J=8.0 Hz), 4.6 (m, 1H), 5.05-5.27 (m, 4H), 7.15 (d, 1H,J=8.7 Hz), 7.46 (d, 1H, J=8.2 Hz); ¹³C NMR(CDCl₃) δ173.6,173.3,172.8,172.1, 169.6, 169.2, 101.5, 74.8, 70.9, 70.8, 69.3,60.5, 53.2, 51.5, 46.1, 41.9, 41.5, 41.0, 39.2, 34.5, 34.3, 34.1, 31.9,31.4, 29.8, 29.6, 29.4, 25.6, 25.3, 25.1, 25.1, 25.0, 24.8, 22.7, 22.5,14.1, 11.1, 8.7.

[0320] Anal. Calcd. for C₈₃H₁₅₈N₃O₁₉P H₂O: C, 64.27; H, 10.40; N, 2.71;P, 2.00. Found: C, 64.14; H, 10.33; N, 2.70; P, 2.05.

EXAMPLE 36 (B36) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₃=N—C₉H₁₉CO, R₂=N—C₅H₁₁CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0321] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 157-158° C.; IR (film)3306, 2955, 2924, 2853, 1734, 1657,1545, 1466, 1378, 1245, 1170, 1081, 954, 842, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (m, 18H),1.15-1.7 (mH),2.2-2.75 (m, 12H),3.06 (q,6H, J=7.2 Hz), 3.36 (d, 1H, J=9.8 Hz), 3.43-3.63 (mH), 3.68-3.95 (m,4H), 4.13-4.27 (m, 2H), 4.54 (d, 1H, J=8.3 Hz), 4.6 (m, 1H),5.08-5.27(m, 4H); ¹³C NMR (CDCl₃) δ 176.9, 173.4, 173.2, 172.8, 172.2, 169.5,169.1, 101.4, 74.8, 71.1, 70.9, 70.8, 69.3, 53.2, 51.6, 46.1, 41.8,41.4, 41.0, 39.2, 34.5, 34.4, 34.3, 34.1, 34.0, 32.0, 31.4, 29.8, 29.6,29.4, 29.3, 25.6, 25.3, 25.2, 25.1, 24.8, 22.7, 22.4, 14.1, 14.0, 8.7.

[0322] Anal. Calcd. for C₈₃H₁₅₈N₃O₁₉P H₂O: C, 64.27; H, 10.40; N, 2.71;P, 2.00. Found: C, 64.09; H, 10.31; N, 2.70; P, 2.06.

EXAMPLE 37 (B37) Preparation ofN-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₂=N—C₉H₁₉CO, R₃=N—C₅H₁₁CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0323] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 156-157° C. dec; IR (film) 3306, 2956, 2923, 2852, 1732, 1659,1545, 1466, 1378, 1246, 1173, 1081, 958, 847, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.0-1.7 (mH), 2.2-2.75 (m, 12H), 2.9-3.3(mH), 3.06 (q, overlaps preceding multiplet, J=7.2 Hz), 3.36 (d, 1H,J=9.6 Hz), 3.43-3.63 (mH), 3.63-3.95 (m, 4H), 4.21 (m, 2H), 4.53 (d, 1H,J=8.0 Hz), 4.6 (br s, 1H), 5.06-5.28 (m, 4H); ¹³C NMR (CDCl₃) δ 176.6,173.6, 173.3, 172.8, 172.1, 169.6, 169.2, 101.5, 74.8, 70.9, 70.9, 69.4,60.5, 53.2, 51.5, 46.1, 41.9, 41.5, 41.1, 39.2, 34.6, 34.5, 34.4, 34.1,31.9, 31.3, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 25.6, 25.3, 25.2, 24.7,22.7, 22.4, 14.1, 14.0, 11.1, 8.7.

[0324] Anal. Calcd. for C₈₃H₁₅₈N₃O₁₉P·H₂O: C, 64.27; H, 10.40; N, 2.71.Found: C, 64.29; H, 10.30; N, 2.61.

EXAMPLE 38 (B38)) Preparation ofN-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=N—C₉H₁₉CO, R₂=R₃=N—C₅H₁₁CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0325] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 152-153° C. dec; IR (film) 3307, 2956, 2924, 2853, 1734, 1658,1544, 1466, 1378, 1316, 1245, 1173, 1081, 955, 843, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.15-1.7 (mH), 2.2-2.75 (m, 12H), 3.06(q, 6H, J=7.2 Hz), 3.28-3.55 (mH), 3.67-3.97 (m, 4H), 4.13-4.27 (m, 2H),4.55 (d, 2H, J=7.2 Hz), 4.60 (m, 1H), 5.08-5.28 (m, 4H), 7.11 (d, 1H,J=8.7 Hz), 7.42 (d, 1H, J=8.0 Hz); ¹³C NMR (CDCl₃) δ 176.9, 173.5,173.2, 172.8, 172.2, 169.5, 169.1, 101.4, 74.8, 71.1, 70.9, 70.8, 69.3,60.5, 53.2, 51.5, 46.1, 41.8, 41.4, 41.1, 39.2, 34.5, 34.3, 34.2, 34.1,34.0, 31.9, 31.7, 31.4, 31.3, 29.8, 29.6, 29.4, 29.3, 25.6, 25.3, 25.2,24.7, 22.7, 22.4, 14.1, 14.0, 11.1, 8.7.

[0326] Anal. Calcd. for C₇₉H₁₅₀N₃O₁₉P: C, 64.24; H, 10.24; N, 2.85.Found: C, 64.06; H, 10.35; N, 2.88.

EXAMPLE 39 (B39) Preparation ofN-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₃=N—C₅H₁₁CO, R₂=N—C₉H₁₉CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0327] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-hexanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 151-152° C. dec; IR (film) 3308, 2956, 2924, 2854, 1732, 1660,1544, 1466, 1378, 1317, 1246, 1173, 1081, 957, 843, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.0-1.7 (mH), 2.18-2.72 (m, 12H), 3.06(q, 6H, J=7.4 Hz), 3.23-3.51 (mH), 3.66-3.98 (m, 4H), 4.12-4.28 (m, 2H),4.55 (d, 1H, J=7.4 Hz), 4.60 (m, 1H), 5.05-5.28 (m, 4H), 7.10 (d, 1H,J=8.2 Hz), 7.43 (d, 1H, J=8.5 Hz); ¹³C NMR(CDCl₃) δ 176.9, 173.6, 173.2,172.7, 172.2, 169.5, 169.0, 101.5, 75.0, 74.8, 71.2, 70.9, 70.8, 69.2,60.5, 53.1, 51.5, 46.1, 41.8, 41.5, 41.1, 39.1, 34.6, 34.5, 34.2, 34.0,32.0, 31.4, 31.3, 29.8, 29.7, 29.6, 29.4, 29.3, 25.6, 25.3, 25.1, 24.8,24.7, 22.7, 22.5, 22.4, 14.1, 14.0, 11.1, 8.7.

[0328] Anal. Calcd. for C₇₉H₁₅₀N₃O₁₉P·H₂O: C, 63.47; H, 10.25; N, 2.81.Found: C, 63.63; H, 10.35; N, 2.84.

EXAMPLE 40 (B40) Preparation ofN-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=R₃=N—C₅H₁₁CO, R₂=N—C₉H₁₉CO,X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0329] In the same manner as described in Example 16 and cognate steps,N-[(R)-3-decanoyloxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-hexanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from 2-(trimethylsilyl)ethyl2-deoxy-4,6-O-isopropylidine-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranoside,L-serine benzyl ester, and (R)-3-hexa- and decanoyloxytetradecanoicacids: mp 158-159° C.; IR (film) 3308, 2956, 2924, 2854, 1734, 1659,1545, 1466, 1378, 1316, 1245, 1173, 1081, 956, 844, 722 cm⁻¹; ¹H NMR(CDCl₃—CD₃OD) δ 0.8-1.0 (m, 18H), 1.15-1.73 (mH), 2.18-2.72 (m, 12H),3.06 (q, 6H, J=7.4 Hz), 3.35 (d, 1H, J=10 Hz), 3.47-3.67 (mH), 3.68-3.97(m, 4H), 4.1-4.3 (m, 2H), 4.54 (d, 1H, J=8.0 Hz), 4.61 (m, 1H),5.07-5.28 (m, 4H); ¹³C NMR (CDCl₃) δ 176.9, 173.5, 173.2, 172.8, 172.2,169.6, 169.1, 101.5, 75.0, 74.8, 71.2, 70.9, 70.8, 69.2, 60.5, 53.2,51.4, 46.1, 41.9, 41.5, 41.0, 39.2, 34.5, 34.2, 34.0, 31.9, 31.4, 29.8,29.6, 29.4, 29.2, 25.6, 25.3, 25.1, 25.0, 24.8, 24.7, 22.7, 22.5, 22.4,14.1, 14.0, 11.1, 8.7.

[0330] Anal. Calcd. for C₇₉H₁₅₀N₃O₁₉P·H₂O: C, 63.47; H, 10.25; N, 2.81;P, 2.07. Found: C, 63.43; H, 10.22; N, 2.83; P, 2.13.

EXAMPLE 41 (B41) Preparation of3-hydroxy-(R)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosideTriethylammonium Salt (Compound (I), R₁=R₃=N—C₉H₁₉CO, R₂=N—C₅H₁₁CO,X=Y=O, N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=OH, P=1 R₈=PO₃H₂).

[0331] In the same manner as described in Example 6 and cognate steps,3-hydroxy-(R)-2-[(R)-3-decanoyloxytetradecanoylamino]propyl2-deoxy-4-O-phosphono-2-[(R)-3-hexanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosidetriethylammonium salt was prepared fromN-(2,2,2-trichloroethoxycarbonylamino)-1,3,4,6-tetra-O-acetyl-2-deoxy-α-D-glucopyranoside,(S)-2-amino-3-benzyloxy-1-propanol, and (R)-3-hexa- anddecanoyloxytetradecanoic acids: mp 151-153° C.; IR (film) 3287, 2956,2923, 2853, 1732, 1643, 1552, 1466, 1378, 1318, 1147, 1176, 1108, 1082,1052, 856, 722 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ0.88 (t, 18H, J=6.9 Hz),1.0-1.72 (mH), 2.17-2.71 (m, 12H), 2.9-3.3 (mH), 3.08 (q, overlapspreceding multiplet, J=7.2 Hz), 3.31 (d, 1H, J=9.6 Hz), 3.5-4.02 (m,8H), 4.20 (d, 1H, J=9.5 Hz), 4.60 (d, 1H, J=8.0 Hz), 5.05-5.25 (m, 4H);¹³C NMR (CDCl₃) 6173.7, 173.5, 173.4, 170.6, 170.1, 101.1, 75.5, 73.0,71.6, 71.3, 70.8, 70.5, 68.2, 61.4, 60.7, 54.8, 50.5, 45.8, 41.4, 39.4,34.6, 34.5, 34.2, 31.9, 31.4, 29.8, 29.7, 29.5, 29.4, 29.3, 25.4, 25.1,22.7, 22.4, 14.1, 14.0, 8.6.

[0332] Anal. Calcd. for C₈₃H₁₆₀N₃O₁₈P·H₂O: C, 64.84; H, 10.62; N, 2.55.Found: C, 65.01; H, 10.50; N, 2.55.

EXAMPLE 42 (B42) Preparation of5-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyloxy]-(S)-4-[(R)-3-decanoyloxytetradecanoylamino]pentanoicAcid Triethylammonium Salt (Compound (I), R₁=R₂=R₃=N—C₉H₁₉CO, X=Y=O,N=M=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, P=2, R₈—PO₃H₂).

[0333] (1) In the same manner described in Example 13-(5), benzyl(S)-4-(t-butyloxycarbonylamino)-5-hydroxypentanoate (0.338 g, 0.954mmol) and the compound prepared in Example 15-(4) (1.15 g, 0.954 mmol)were coupled in the presence of AgOTf (1.22 g, 4.77 mmol) to give 0.70 g(50%) of benzyl(S)-4-(t-butyloxycarbonylamino)-5-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosyloxy]pentanoate:¹H NMR (CDCl₃) δ 0.88 (m, 6H), 1.0-2.05 (m, 55H), 2.12-2.5 (m, 6H),3.28-3.90 (m, 5H), 4.26 (dd, 1H, J=4.5, 11.5 Hz), 4.38 (d, 1H, J=11.5Hz), 4.57-4.98(m, 5H),5.11 (s, 2H),5.18(m, 1H), 5.49 (t, 1H, J=9Hz),5.78(d, 1H, J=7.7 Hz), 7.04-7.45 (m, 15H).

[0334] (2) A solution of the compound prepared in (1) above (0.67 g,0.45 mmol) in CH₂Cl₂ (5 mL) was cooled to 0° C., treated dropwise withTFA (70 μL), and stirred for 3 h at room temperature. The reactionmixture was diluted with CH₂Cl₂ (15 mL), washed with saturated aqueousNaHCO₃ and dried (Na₂SO₄). (R)-3-Decanoyloxytetradecanoic acid (0.20 g,0.50 mmol) and EDC MeI (0.15 g, 0.5 mmol) were added and the resultingmixture was stirred for 16 h at room temperature. The reaction mixturewas filtered through a pad of Celite® and concentrated. The crudeproduct obtained was purified by flash chromatography on silica gel(gradient elution, 15→30% EtOAc-hexanes) to give 0.36 g (45%) of benzyl(S)-4-[(R)-3-decanoyloxytetradecanoylamino]-5-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-decanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosyloxy]pentanoate:¹H NMR (CDCl₃) δ 0.89 (m, 12H), 1.0-1.978 (n1H), 2.12-2.5 (m, 10H),3.45-3.65 (m, 2H), 3.79 (dd, 2H, J=3.8, 10 Hz), 4.06 (m, 1H), 4.27 (dd,1H, J=4.9, 12 Hz), 4.35 (d, 1H, J=12 Hz), 4.6-4.8 (m, 3H), 4.83 (d, 1H,J=8.3 Hz), 5.10 (s, 2H), 5.17 (m, 2H), 5.48 (t, 1H, J=10 Hz), 5.79 (d,1H, J=7.7 Hz), 6.05, (d, 1H, J=8.8 Hz), 7.07-7.42 (m, 15H).

[0335] (3) In the same manner as described for the preparation ofcompound B14 from the compound prepared in Example 15-(5),5-[2-deoxy-4-O-phosphono-2-[(R)-3-decanoyloxytetradecanoylamino]-3-O-[(R)-3-decanoyloxytetradecanoyl]-α-D-glucopyranosyloxy]-(S)-4-[(R)-3-decanoyloxytetradecanoylamino]pentanoicacid triethylammonium salt was prepared from the compound prepared in(2) above: Mp 184-188° C.; IR (film) 3284, 2955, 2919, 2848, 1730, 1654,1548, 1459, 1374, 1259, 1165, 1081, 1032, 800 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD)δ 0.88 (t, 18H, J=7 Hz), 1.0-2.0 (mH), 2.18-2.75 (m, 12H), 3.08 (q, 6H,J=7.4 Hz), 3.33-4.42 (mH), 4.44 (d, 1H, J=8.5 Hz), 5.02-5.31 (m,4H),7.54(d, 1H, J=8 Hz),),7.61 (d, 1H, J=7 Hz); ¹³C NMR(CDCl₃) δ 176.8,173.6, 173.3, 170.8, 170.2, 101.1, 75.1, 73.7, 71.7, 71.1, 70.8, 70.1,60.8, 54.1, 48.8, 45.9, 41.4, 41.2, 39.4, 34.5, 34.4, 34.1, 31.9, 31.3,29.8, 29.7, 29.6, 29.5, 29.4, 26.9, 25.5, 25.3, 25.1, 22.7, 14.1, 8.6.

[0336] Negative FAB-MS calcd for [M−H]⁻1514.0889, found 1514.0816.

EXAMPLE 43 (B43) Preparation ofN-[(R)-3-hydroxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serineTriethylammonium Salt. (Compound (I), R₁=N—C₁₃H₂₇CO, R₂=N—C₁₁H₂₃CO,R₃=H, X=Y=O, N=M=P=Q=0, R₄=R₅=R₇=R₉=H, R₆=CO₂H, R₈=PO₃H₂).

[0337] (1) In the same manner described in Example 13-(5),N-allyloxycarbonyl-L-serine benzyl ester (0.225 g, 0.806 mmol) and thecompound prepared in Example 22-(2) (1.104 g, 0.886 mmol) were coupledin the presence of AgOTf (0.828 g, 3.22 mmol) to give 1.01 g (83%) ofN-allyloxycarbonyl-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (m, 6H), 1.0-1.7 (m, 42H), 1.78 (s,3H), 1.86 (s, 3H), 2.12-2.48 (m, 4H), 3.26 (m, 1H), 3.66 (m, 1H), 3.80(dd, 1H, J=3, 10 Hz), 4.19-4.38 (m, 4H), 4.48-4.85 (m, 6H), 4.98(d, 1H,J=7.7 Hz),5.08-5.38(m, 5H),5.49(m, 1H),5.60-5.75(m, 2H),5.82-6.0 (m,1H), 7.06-7.42 (m, 15H).

[0338] (2) A solution of the compound prepared in (1) above (1.01 g,0.68 mmol) and diethyl malonate (1.50 g, 9.48 mmol) in THF was degassedwith argon (1 h), treated with tetrakis(triphenylphosphine)palladium(0)(0.10 g, 0.09 mmol), and stirred overnight at room temperature. Thereaction mixture was filtered through a pad of silica with 2% MeOH—CHCl₃and the filtrate concentrated. A solution of the crude amine obtained inCH₂Cl₂ (20 mL) was treated with (R)-3-hydroxytetradecanoic acid (0.18 g,0.75 mmol) and EDC·MeI (0.66 g, 1.02 mmol), stirred overnight at roomtemperature, and then concentrated. The crude product obtained waspurified by flash chromatography on silica gel (gradient elution, 30→40%EtOAc-hexanes) to give 0.506 g (46%) ofN-[(R)-3-hydroxytetradecanoyl]-O-[2-deoxy-4-O-diphenylphosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-6-O-(2,2,2-trichloro-1,1-dimethylethoxycarbonyl)-2-(2,2,2-trichloroethoxycarbonylamino)-α-D-glucopyranosyl]-L-serinebenzyl ester: ¹H NMR (CDCl₃) δ 0.88 (m, 9H), 1.0-1.7 (m, 62H), 1.79 (s,3H), 1.87 (s, 3H), 2.19 (t, 2H, J=7 Hz), 2.3-2.5 (m, 4H), 3.1 (br s,1H), 3.55 (q, 1H, J=9 Hz), 4.0-4.43 (m, 5H), 4.56-4.85 (m, 4H),5.13-5.32 (m, 4H), 6.59 (d, 1H, J=7.4 Hz), 6.83 (br s, 1H), 7.17-7.41(m, 15H).

[0339] (3) In the same manner as described for the preparation ofcompound B12 from the compound prepared in Example 13-(5),N-[(R)-3-hydroxytetradecanoyl]-O-[2-deoxy-4-O-phosphono-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-α-D-glucopyranosyl]-L-serinetriethylammonium salt was prepared from the compound prepared in (2)above: mp 170-173° C. dec; IR (film) 3313, 2955, 2923, 2853, 1734, 1662,1655, 1558, 1541, 1467, 1458, 1376, 1248, 1166, 1108, 1078, 1049, 953,942, 842 cm⁻¹; ¹H NMR (CDCl₃—CD₃OD) δ 0.88 (m, 18H), 1.15-1.7 (mH),2.2-2.75(m, 12H), 3.06 (q, 6H, J=7.2 Hz), 3.3-3.63 (mH), 3.66-3.98 (m,4H), 4.1-4.3 (m, 2 H), 4.54 (d, 1H, J=8.0 Hz), 4.6 (m, 1H), 5.05-5.27(m, 4H), 7.15 (d, 1H, J=8.7 Hz), 7.46 (d, 1H, J=8.2 Hz); ¹³C NMR(CDCl₃)δ 177.0, 173.3, 172.8, 172.3, 171.9, 169.2, 101.2, 74.9, 74.8, 74.3,70.8, 70.6, 69.3, 68.4, 59.9, 53.1, 51.5, 42.5, 41.5, 39.2, 37.1, 34.6,34.4, 34.3, 34.1, 32.0, 29.8, 29.4, 25.6, 25.2, 22.7, 22.5, 14.5, 8.7.

[0340] Anal. Calcd. for C₈₃H₁₆₀N₃O₁₈P 2H₂O: C, 64.10; H, 10.63; N, 2.70;P, 1.99. Found: C, 64.28; H, 10.42; N, 2.70; P, 1.84.

TEST EXAMPLE 1 Stimulation of Anti-Tetanus Toxoid Antibody Production

[0341] The AGPs of the subject invention enhanced antibody production topurified tetanus toxoid in a murine model. Ten mg of each AGP sample wasadded to 1 ml of an oil-lecithin mixture containing squalene oil plus12% lecithin. The mixtures were heated in a 56° C. water bath andsonicated to achieve clear solutions. Fifty (50) μl of each solution wasemulsified by vortexing in 2 ml of sterile, pre-warmed 0.1% Tween 80saline containing 1.0 μg tetanus toxoid antigen/ml. Preparations werevortexed again just prior to administration to mice. Female C57BL/6×DBA/2 F₁ mice (8 per group) were treated with 0.2 ml of the appropriatepreparation distributed as a 0.1 ml subcutaneous injection into eachflank. The final mouse dosage of the tetanus toxoid and AGP compoundswas 0.2 μg and 50 μg, respectively. Control mice received tetanus toxoidin vehicle (oil-Tween saline). All mice were treated on day 0 followedby a second immunization on day 21. Fourteen days following the secondimmunization mice were bled and sera were isolated by centrifugation.

[0342] Serum samples from each mouse were evaluated for anti-tetanustoxoid antibodies by enzyme immunoassay (EIA) analysis using tetanustoxoid coated microtiter plates. Anti-tetanus antibody titers wereevaluated for IgM, total Ig, as well as, IgG₁, IgG_(2a) and IgG_(2b)isotypes. Each serum sample was diluted 2-fold for eleven dilutionsstarting with an initial serum dilution of 1:200. Results are shown inTables 2-4. TABLE 2 Anti-tetanus toxoid antibody titers of treated mice.Total IgG IgG₁ IgG_(2a) IgG_(2b) IgM Material T/C* Titer T/C Titer T/CTiter T/C Titer T/C Titer B11 3.6 23,200 1.86 400,000 2.06 10,450 0.9326,800 4.75 7,600 B2 3.84 24,800 2.16 464,000 4.28 21,700 1.57 45,2004.50 7,200 B1 3.97 25,600 3.42 736,000 3.78 19,200 2.45 70,400 2.383,800 B25 8.93 57,600 2.68 576,000 1.67 8,500 3.28 94,400 2.0 3,200 B214.71 30,400 2.23 480,000 5.83 29,600 6.07 174,400 5.50 8,800 B15 18.85121,600 4.17 896,000 6.80 34,500 2.79 80,256 4.0 6,400 Vehicle 6,450215,000 5,075 28,750 1,600

[0343] TABLE 3 Anti-tetanus toxoid antibody titers of treated mice.Material T/C* IgM T/C IgG_(2a) T/C IgG_(2b) B12 3.1 4800 139.4 2370 1499840 B16 1.6 2560 66.8 1135 104 6880 B13 3.9 6080 220 3740 >208 >13,760B11 3.3 5120 347 5900 127.3 8400 Vehicle — 1760 — 25 — 98

[0344] TABLE 4 Anti-tetanus toxoid antibody titers of treated mice.Total Ig IgM IgG₁ IgG_(2a) IgG_(2b) Material T/C Titer T/C Titer T/CTiter T/C Titer T/C Titer B26 10.5 2,490 1.1 600 16.9 25,200 29.3 44042.6 2,260 B15 144.5 34,400 2.7 1,520 118.3 176,000 259.3 3,890 603.832,000 B22 60.0 19,050 0.8 440 18.4 27,400 345.8 5,187 59.6 3,160 B28228.6 54,500 3.7 2,080 92.5 137,600 664.7 9,970 519.2 27,520 Vehicle 238560 1,488 15 53

[0345] Compounds of the subject invention showed a dose response whenadministered with tetanus toxoid. BFD 1 (C57B1/6X DBA/2) female mice (8per group) were immunized with 0.2 ml of emulsions containing AGP+0.2 μgof tetanus toxoid. A second immunization was administered 21 days postprimary immunization. Each mouse was bled 21 days after the secondinjection. The results are shown in Tables 5 and 6. TABLE 5 Doseresponse of AGPs in mice immunized with tetanus toxoid. Total Ig IgMIgG₁ IgG_(2a) IgG_(2b) T/C T/C T/C T/C T/C Material Ratio* Titer RatioTiter Ratio Titer Ratio Titer Ratio Titer B15 50 μg 3.3 7,000 13.437,600 4.1 26,300 150.0 11,225 3.2 2500 B15 25 μg 5.8 12,400 2.1 6,0004.5 28,800 52.0 3900 7.0 5400 B15 10 μg 5.3 11,450 1.4 4,000 5.5 35,10033.8 2538 9.9 7650 B27 50 μg 3.2 6,800 4.0 11,200 1.6 10,400 12.0 90011.6 9,000 Vehicle 2150 2800 6350 75 775

[0346] TABLE 6 Dose response of AGPs in mice immunized with tetanustoxoid. IgM Total Ig IgG₁ IgG_(2a) IgG_(2b) Material T/C* Titer T/CTiter T/C Titer T/C Titer T/C Titer B12 50 μg 5.43 869 368.55 47,543141.22 259,429 nd 499.35 12,983 B12 25 μg 3.14 503 403.98 52,114 145.21266,743 16.86 354 196.92 5,120 B12 10 μg 3.71 594 248.06 32,000 81.12149,029 6.81 143 181.12 4,709 B12 5 μg 3.43 549 489.92 63,200 84.11154,514 34.14 717 352.54 9,166 B12 1 μg 1.71 274 326.02 42,057 90.08165,486 73.71 1,548 175.81 4,571 B15 50 μg 3.14 503 233.88 30,171 90.08165,486 50.05 1,051 235.62 6,126 B15 25 μg 2.29 366 181.91 23,467 106.14194,971 10.43 219 158.23 4,114 B15 10 μg 2.86 457 170.10 21,943 39.0771,771 2.57 54 84.38 2,194 B15 5 μg 1.71 274 248.06 32,000 103.15189,486 3.00 63 210.88 5,483 B15 1 μg 1.57 251 166.56 21,486 72.04132,343 7.62 160 114.27 2,971 Vehicle 160 129 1837 21 26

TEST EXAMPLE 2 Stimulation of Antiovalbumin Antibody Production

[0347] BDF1 female mice (8 per group) were immunized with 0.2 ml ofemulsions containing 50 μg of the AGPs +50 μg of ovalbumin. A secondimmunization was administered 21 days post primary. Each mouse was bled14 days after the second injection. Antibody titers of immunized miceshowing total IgG and IgM as well as titers for the subgroups of IgGincluding IgG₁, IgG_(2a) and IgG_(2b) are given in Table 7. TABLE 7Adjuvant activity in BDF1 mice immunized with ovalbumin. Total Ig IgMIgG1 IgG2a IgG2b Material T/C* Titer T/C Titer T/C* Titer T/C Titer T/CTiter B11 0.7 150 1.3 250 1.6 2650 1.7 550 1.6 375 B2 2.5 563 0.9 1755.0 8300 2.5 825 2.3 550 B1 0.5 119 0.8 150 0.5 763 0.2 56 0.8 188 B251.9 438 0.8 150 5.2 8500 0.5 163 5.0 1188 B21 0.5 113 1.3 250 0.6 10000.1 25 0.8 200 B15 4.1 925 2.3 438 0.6 950 0.3 113 16.7 3963 B27 0.6 1381.6 300 0.8 1275 0.1 38 0.5 113 Vehicle — 225 — 188 — 1650 — 325 — 238

[0348] The AGP compounds of the subject invention when administered to awarm-blooded animal with the antigen ovalbumin stimulates the productionof antibody to that antigen.

TEST EXAMPLE 3 Generation of a Protective Immune Response to InfectiousInfluenza

[0349] Mice vaccinated with formalin-inactivated influenza and the AGPcompounds of the subject invention mounted a protective immune responseto an influenza challenge as well as produced antibody to that antigen.Animals were vaccinated with the antigen and AGP compounds in variouscarriers. The degree of protection was determined by challenging themice with intranasal (IN) administration of approximately 10 LD₅₀infectious influenza A/HK/68. Mortality was assessed for 21 daysfollowing the challenge. The number of mice surviving the challenge doseis a direct assessment of the efficacy of the vaccine. For theexperiments provided this data does not necessarily correlate with theamount of antibody produced.

[0350] 1) Vaccines were formulated in 0.2% triethanolamine (TEoA)/watersolution containing: 1 hemagglutinating unit (HAU) offormalin-inactivated influenza A/HK/68 (FI-Flu), and 50 μg of AGP exceptthe vehicle control vaccines which contained no AGP. ICR mice (10/group)were vaccinated 1 time only. The vaccines were administered bysubcutaneous (SQ) injection of 0.1 ml/site at 2 distinct sites near theinguinal lymph nodes for a total of 0.2 ml of vaccine/mouse. Mice (only5 mice/group) were bled from the orbital plexus 14 days following thevaccination. Sera was harvested and frozen at −20° C. until used forenzyme-linked immunosorbent assay (ELISA). All mice were challenged 30days post vaccination by intranasal (IN) administration of approximately10 LD₅₀ infectious influenza A/HK/68. Mortality was assessed for 21 daysfollowing the challenge. Anti-influenza antibody titers obtained fromvaccinations with TEoA formulations and corresponding survival rates ofmice vaccinated with this formulation are shown in Table 8. TABLE 8Anti-influenza antibody titers and survival rates of treated mice.Titer⁻¹ Material Total IgG Percent Survival Nonimmune <100 0 Vehicle<100 0 B9 6400 44 B10 1600 40 B7 200 33 B3 1600 33 B14 6400 44 B15 640050

[0351] 2) Vaccines were formulated in 2% Squalene solution containing: 1hemagglutinating unit (HAU) of formalin-inactivated influenza A/HK/68(FI-Flu), and 25 μg of AGP except the saline and vehicle controlvaccines which contained no AGP. BALB/c mice (10/group) were vaccinated1 time only. The vaccines were administered by subcutaneous (SQ)injection of 0.1 ml/site at 2 distinct sites near the inguinal lymphnodes for a total of 0.2 ml of vaccine/mouse. Mice (only 5 mice/group)were bled from the orbital plexus 14 days following the vaccination.Sera was harvested and frozen at −20° C. until used for enzyme-linkedimmunosorbent assay (ELISA). All mice were challenged 35 days postvaccination by intranasal (IN) administration of approximately 10 LD₅₀infectious influenza A/HK/68. Mortality was assessed for 21 daysfollowing the challenge. Anti-influenza antibody titers obtained fromvaccinations with the squalene formulations as well as correspondingsurvival rates of vaccinated animals are shown in Table 9. TABLE 9Anti-influenza antibody titers and survival rates of treated mice.Titer⁻¹ Percent Material Total IgG IgG1 IgG2a IgG2b Survival Nonimmune<100 <100 <100 <100 0 Saline 800 100 800 100 62.5 Vehicle 1600 1600 16001600 100 B25 3200 1600 6400 1600 100 B15 1600 3200 3200 400 100 B9 16001600 3200 800 87.5 B10 400 400 400 400 62.5 B3 3200 3200 6400 800 87.5B6 800 800 400 1600 75 B14 3200 6400 3200 6400 87.5 B28 800 400 400 10050

[0352] 3) The antibody titers and survival rate of vaccinated mice werecompared after a primary then a secondary vaccination. Vaccines wereformulated in 0.2% TEoA/water solution containing: 1 hemagglutinatingunit of formalin-inactivated influenza A/HK/68, 25 μg AGP, except thevehicle control vaccine that contained no AGP. ICR mice (20/group) wereadministered vaccines by subcutaneous injection of 0.1 ml/site at 2distinct sites near the inguinal lymph nodes for a total of 0.2 ml ofvaccine/mouse. Each group was split into 2 subgroups 35 days after theprimary vaccination. One of each subgroup was challenged at this time,the remaining subgroups received a secondary vaccination. Mice (only5/subgroup) were bled from the orbital plexus 14 days followingvaccination (primary or secondary). Sera was harvested and frozen at−20° C. until used for ELISA. Mice were challenged 35 post primary, orsecondary, vaccination by intranasal administration of approximately 10LD50, or 40 LD50, infectious influenza A/HK/68, respectively. Mortalitywas assessed for 21 days following the challenge. Anti-influenzaantibody titers and survival rates of mice post primary and postsecondary vaccination are shown in Table 10. Antibody titers as well assurvival rates of mice vaccinated a second time were higher. TABLE 10Antibody titers and survival rates of treated mice. IgG Titer-1 PercentSurvival Material post 1° post 2° post 1° post 2° Nonimmune 200 100 0 0Vehicle 800 102,400 20 40 B9 6400 12,800 80 50 B10 1600 25,600 60 90 B73200 >102,400 60 60 B4 800 25,600 50 70 B3 3200 102,400 70 60 B51600 >102,400 60 90 B6 1600 102,400 80 70 B14 800 51,200 33 70

TEST EXAMPLE 4 The Effect of Fatty Acid Chain Length on Adjuvanticity

[0353] The effect of the length of fatty acid chains R₁—R₃ on activitywas tested. Vaccines were formulated in 0.2% TEoA/water solutioncontaining: 1 hemagglutinating unit of formalin-inactivated influenzaA/HK/68, and 25 μg of AGP, except the vehicle control vaccines, whichcontained no AGP. ICR mice (10/group) were vaccinated 1 time only. Thevaccines were administered by subcutaneous injection of 0.1 ml/site at 2distinct sites near the inguinal lymph nodes for a total of 0.2 ml ofvaccine/mouse. Mice (only 5 mice/group) were bled from the orbitalplexus 14 days following the vaccination. Sera was harvested and frozenat −20° C. until used for ELISA. All mice were challenged 35 postvaccination by intranasal administration of approximately 10 LD₅₀infectious influenza A/HK/68. Mortality was assessed for 21 daysfollowing the challenge. The length of the fatty acid chain appears tomildly affect biological activity. Results are shown in Tables 11 and12. TABLE 11 Antibody titers and survival rates of treated mice. ChainTiter⁻¹ Percent Material Length Total IgG IgG1 IgG2a IgG2b SurvivalNonimmune — 200 100 100 800 0 Vehicle — 200 100 100 200 11 B18 7 800 800800 400 20 B17 8 6400 3200 3200 1600 40 B16 9 800 1600 100 800 40 B15 103200 200 3200 6400 70 B14 10 800 1600 100 400 30 B13 11 1600 800 400 80050 B12 12 200 200 100 200 0 B11 14 1600 200 1600 400 30

[0354] TABLE 12 Antibody titers and survival rates of treated mice.Chain Titer⁻¹ Percent Material Length Total IgG IgG1 IgG2a IgG2bSurvival Nonimmune — 100 100 50 800 0 Vehicle — 100 200 50 100 30 B8 76400 3200 400 1600 80 B7 9 3200 3200 100 1600 70 B5 10 800 200 50 400 44B4 11 3200 400 100 1600 60 B3 12 1600 1600 50 800 0 B1 14 12,800 64001600 15600 40

TEST EXAMPLE 5 The Effect of Variations in the Carbon Chain LengthBetween the Heteroatom X and the Aglycon Nitrogen Atom on Adjuvanticity

[0355] The length of the carbon chain between X and the aglycon nitrogenatom was extended progressively by a single atom. The effect oflengthening the chain between these two components on adjuvanticity wasexplored. Vaccines were formulated in 0.2% TEoA/water solutioncontaining: 1 hemagglutinating unit of formalin-inactivated influenzaA/HK/68, and 25 μg of AGP, except the vehicle control vaccines, whichcontained no AGP. ICR mice (10/group) were vaccinated 1 time only. Thevaccines were administered by subcutaneous injection of 0.1 ml/site at 2distinct sites near the inguinal lymph nodes for a total of 0.2 ml ofvaccine/mouse. Mice (only 5 mice/group) were bled from the orbitalplexus 14 days following the vaccination. Sera was harvested and frozenat −20° C. until used for ELISA. All mice were challenged 35 days postvaccination by intranasal administration of approximately 10 LD₅₀infectious influenza A/HK/68. Mortality was assessed for 21 daysfollowing the challenge. Adjuvant activity appears to lessen as thelength of the carbon chain between the heteroatom X and aglycon nitrogenatom increases. However, depending upon the residues attached to thiscarbon chain the biologic and metabolic stability of the molecules maybe affected. Results are shown in Tables 13. TABLE 13 Antibody titersand survival rates of treated mice. Carbon Titer−1 Percent MaterialChain Total IgG IgG1 IgG2a IgG2b Survival Nonimmune — <50 <50 <50 <50 0Vehicle — 200 200 50 200 25 B19 2 12,800 100 800 6400 50 B21 3 6400 800100 1600 40 B22 4 3200 100 3200 200 40

TEST EXAMPLE 6 Cytokine Induction by the AGP Compounds

[0356] The AGP compounds of the subject invention induced cytokines inhuman whole blood ex vivo culture assays. AGP compounds were solubilizedin 10% EtOH-water and diluted to various concentrations. Fifty μl ofeach dilution were added to 450 μl of whole human blood. Controls weretreated with culture media (RPMI). The reaction mixture was incubated at37° C. for 4 hr with constant mixing on a rotator. Sterile PBS (1.5 ml)was added to the reaction mixture, the cells were centrifuged and thesupernatents removed for cytokine testing. The concentration of TNF-αand IL-1β in each supernatent was determined using immunoassay ELISAkits from R&D Systems. Results from these studies are shown in Tables14-19. TABLE 14 Stimulation of cytokine secretion in an ex vivo assay.Dosage TNF-α IL-1β Material (μg) (pg/ml) (pg/ml) B26 20 498.90 33.25 10254.94 25.34 5 75.62 9.89 1 38.85 3.90 B2 20 1338.42 155.07 10 817.67114.41 5 235.32 34.72 1 105.52 14.53 RPMI — 2 0

[0357] TABLE 15 Stimulation of cytokines in an ex vivo assay. DosageTNF-α IL-1β Material (ng/ml) (pg/ml) (pg/ml) B16 10,000 291 55 5000 27753 1000 155 39 B13 10,000 775 THTC* 5000 716 187 1000 740 177 B9 10,000449 96 5000 247 84 1000 145 53 B10 10,000 207 43 5000 127 61 1000 73 17B7 10,000 83 16 5000 57 14 1000 26 6 RPMI — 2 0

[0358] TABLE 16 Stimulation of cytokines in an ex vivo assay. DosageTNF-α IL-1β Material (ng/ml) (pg/ml) (pg/ml) B4 10,000 432 213 5000 205164 1000 94 70 B3 10,000 567 269 5000 390 342 1000 189 204 B5 10,000 16979 5000 143 162 1000 43 36 B6 10,000 94 52 5000 59 29 1000 30 13 B1410,000 249 91 5000 120 71 1000 56 46 RPMI — 2 0

[0359] TABLE 17 Stimulation of cytokine secretion in an ex vivo assay.Dosage TNF-α IL-1β Material (ng/ml) (pg/ml) (pg/ml) B11 10,000 181 62.35000 139 61.7 1000 115 54.5 500 125 55.8 100 127 59.8 B13 10,000 583 2825000 592 390 1000 478 327 500 411 352 100 302 261 B15 10,000 320 1535000 280 126 1000 209 94.4 500 183 104 100 133 51.6 B16 10,000 121 41.05000 114 34.0 1000 72 19.5 500 55 17.1 B14 10,000 114 24.6 5000 87 19.01000 51 10.0 500 49 19.9 RPMI — 2 0

[0360] TABLE 18 Stimulation of cytokine secretion in an ex vivo assay.Dosage TNF-α IL-1β Material (ng/ml) (pg/ml) (pg/ml) B2 10,000 100 22.25000 75 14.0 1000 38 9.0 500 28 8.3 100 6.1 3.5 B1 10,000 20 10.0 500011 5.5 1000 2.8 4.0 500 1.1 0 100 0 0 B7 10,000 61 14.7 5000 44 8.3 100030 4.3 500 27 3.8 100 10 5.1 B4 10,000 232 66.9 5000 173 66.5 1000 13032.0 500 116 19.3 100 89 65.2 B3 10,000 433 151.9 5000 316 200.4 1000229 75.1 500 212 67.9 100 130 35.9 B5 10,000 142 24.1 5000 99 23.0 100096 10.5 500 59 16.9 100 33 5.4 RPMI — 2 0

[0361] TABLE 19 Stimulation of cytokine secretion in an ex vivo assay.Dosage TNF-α IL-1β Material (ng/ml) (pg/ml) (pg/ml) B17 10,000 2.8 05000 2.2 0 1000 2.6 0.2 B8 10,000 2.8 0 5000 0.7 0.5 1000 1.5 0.1 B2210,000 287 17 5000 11 1.9 1000 2.2 0.1 B28 10,000 198 13 5000 197 131000 139 8 B12 10,000 1017 135 5000 957 153 1000 863 175 RPMI — 3.9 0

TEST EXAMPLE 7 Stimulation of a Cytotoxic T-Lymphocyte Response

[0362] The induction of a cytotoxic T-lymphocyte response afteradministration of the AGP compounds of the subject invention and aprotein antigen was detected by a cytotoxicity assay. Groups of C57BL/6mice were given a primary immunization subcutaneously (inguinal region)with 25 μg ovalbumin (OVA) formulated in AGP preparations. The injectedvolume was 200 μl. Twenty-one days later three mice per experimentalgroup were killed and spleens removed and pooled as single cellsuspensions and counted.

[0363] Spleen cells (75×10⁶ cells in 3-4 ml media) from the experimentalgroups were placed in a 25 cm² T-flask. Next, 1.0 ml of irradiated(20,000 rads) E.G7 (OVA) cells at 5×10⁶/ml were added to the flask. Thevolume was brought to 10 ml. The cultures were maintained by placing theT-flasks upright in a 37° C., 5% CO₂ incubator for four days. On day 4the surviving cells were recovered from the flasks, washed 1× in freshmedia resuspended in 5.0 ml, and counted.

[0364] Recovered effector cells were adjusted to 5×10⁶ viable cells/mland 100 μl volumes were diluted serially in triplicate in wells of 96well round-bottom plates (Coming 25850) using 100 μl/well of media as adiluent. Next, 100 μl volumes of ⁵¹Cr-labelled (see below) targets [E.G7(OVA)-an ovalbumin gene transfected EL-4 cell line] at 1×10⁵ cells/mlwere added to the wells. Spontaneous release (SR) wells contained 100 μlof targets and 100 μl of media. Maximal release (MR) wells contained 100μl of targets and 100 μl detergent (2% Tween 20). Effector/target (E/T)ratios were 50:1, 25:1, 12.5:1. The plates were centrifuged at 400× gand incubated at 37° C., 5% CO₂ for 4 hr. After the incubation the wellsupernatants were collected using a Skatron Supernatant CollectionSystem. Percent specific lysis=$100 \times \left\lbrack \frac{\left( {{{Exp}.{Release}} - {SR}} \right)}{\left( {{MR} - {SR}} \right)} \right\rbrack$

[0365] Target cells, E.G7 (OVA), were labelled with ⁵¹Cr (sodiumchromate) as follows. In a total volume of 10 ml were mixed 5×10⁶ targetcells and 250 μCi ⁵¹Cr in 15 ml conical tube. The cell suspension wasincubated in a 37° C. water bath for 90 min., with gentle mixing every15 min. After incubation the labelled cells were washed 3× bycentrifugation and decanting with 15 ml volumes of media. After thethird centrifugation the cells were resuspended in 10 ml of fresh mediaand allowed to stand at room temperature for 30 min. and thencentrifuged. The cells were finally resuspended in media at 1×10⁵cells/ml.

[0366] Mice immunized according to the procedure above with the AGPs ofthe subject invention displayed a cytotoxic T-lymphocyte response to theOVA antigen as shown in Table 20. TABLE 20 Cytotoxic T-lymphocyteresponse of treated cells. % Cytotoxicity E:T Material 50:1 25:1 12.5:1B11 14 8 5 B12 13 7 4 B13 28 15 10 B15 58 49 30 B16 42 29 20 B17 39 2615 B18 36 20 15 B14 45 36 25 B28 28 15 9 B27 17 9 5 B1 34 24 15 B3 65 5442 B4 72 66 60 B5 28 18 11 B7 57 44 29 B8 36 20 15 B10 65 56 38 B9 65 5536 B6 54 41 37 B2 21 12 6 B25 65 55 43 B26 14 8 4 B22 58 42 31 B21 38 2615 B19 59 42 33 B20 36 25 13 B29 16 9 5 B31 19 11 7 B35 9 5 2 B36 13 7 4B37 12 8 6 B38 38 25 16 B39 33 21 13 B40 20 12 8 B43 19 12 6 VehicleControl <10

TEST EXAMPLE 8 Generation of Serum and Mucosal Antibody Titers toTetanus-Toxoid

[0367] The AGPs of the subject invention elicited both a serum andmucosal immune response to purified tetanus toxoid when administeredintranasally. Groups of BALB/c mice were given a primary immunization(1°) intranasally with 10 μg tetanus toxoid (TT)+20 μg AGP formulated inan aqueous formulation (AF) in a volume of 20 μl. A secondaryimmunization (2°) was given 14 days later and a tertiary immunization(3°) identical in composition to the first and second was administered14 days later. Mice were bled on day 21 (day 7 post 2°) and day 38 (day10 post 3°) and day 48 (day 20 post 3°). Vaginal wash/fecal extractsamples were taken on day 7 post 2° and day 7 post 3°. Serum and washsamples were assayed for anti-TT antibody by standard ELISA methods.Results of these assays are shown in Tables 21 and 22 below.

[0368] The aqueous formulation comprises the AGPs of the subjectinvention and one or more surfactants. Surfactants useful in an aqueouscomposition include glycodeoxycholate, deoxycholate, sphingomyelin,sphingosine, phosphatidylcholine,1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine,L-α-phosphatidylethanolamine, and1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, or a mixture thereof. Theaqueous formulation used in this example comprises the surfactant 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and was prepared asfollows: briefly; a 4 mg/ml solution of DPPC was prepared in ethanol. Analiquot of the ethanol solution is added to the dried AGPs and swirledgently to wet the AGP. The ethanol is removed by blowing a stream offiltered nitrogen gently over the vial. Water for Injection is added andthe suspension is sonicated 10 min. at 60° C. until clear. The resultingaqueous formulation contains approximately 118 μg/ml DPPC, has particlesof around 70 nm and was filter sterilized. TABLE 21 Anti-tetanus toxoidantibody titers in treated mice. Anti-Tetanus Toxoid Titer⁻¹ VaginalWash Fecal Extract IgG IgA IgG IgA Material 2° 3° 2° 3° 2° 3° 2° 3° B25800 6400 6400 6400 50 200 3200 6400 B15 400 800 6400 6400 50 100 640012,800 B19 200 400 1600 3200 25 25 3200 6400 B4 1600 400 1600 6400 25 503200 12,800 B5 3200 800 3200 3200 50 100 3200 6400 B3 1600 1600 64006400 50 100 3200 6400 B22 400 800 800 3200 25 50 1600 6400 PBS <25 <25<25 <25 <25 <25 <25 <25 Normal Sera <25 <25 <25 <25 <25 <25 <25 <25

[0369] TABLE 22 Serum anti-tetanus toxoid antibody titers in treatedanimals. Anti-Tetanus Toxoid Titer⁻¹ Serum Pools IgG₁ IgG_(2a) IgA d21d38 d48 d21 d38 d48 d21 d38 d48 B25  1M* 8M 8M 512K 4M 4M 12.8K 102.4K102.4K B15 2M 8M 8M 512K 1M 2M 12.8K  51.2K  25.6K B19 2M 4M 4M  64K#256K   128K    6.4K  25.6K  12.8K B4 1M 8M 8M   1M 2M 2M 25.6K 102.4K102.4K B5 2M 8M 8M 512K 2M 2M 25.6K 102.4K 102.4K B3 512K   4M 8M 512K2M 2M 12.8K  51.2K  51.2K B22 1M 2M 4M  64K 256K   256K    6.4K  25.6K 25.6K PBS 1,000     16K   16K   1,000   1,000     1,000     200     200    200   C 200    200    200    100   100    100    200      200   200  

[0370] Intranasal administration of TT formulated in AGP-AF induced bothan antigen specific humoral immune response (Table 22) and a mucosalimmune response (Table 21) to that antigen.

TEST EXAMPLE 9 Stimulation of an Immune Response to Hepatitis B SurfaceAntigen by Intranasal Administration

[0371] Mice administered hepatitis B surface antigen (HBsAg)intranasally with the compounds of the subject invention produced serumIgG and IgA titers to that antigen. Secretory IgA was detected invaginal washes and the induction of a cytotoxic T-lymphocyte responsewas detected by a cytotoxicity assay.

[0372] Groups of BALB/c mice were given a primary immunization (1°)intranasally with 2.5 μg HBsAg+10 μg AGP-AF in a volume of 20 μl. AGP-AFwas prepared as in TEST EXAMPLE 8. Twenty-one days later mice were givena secondary immunization (2°) of 7.5 μg HBSAG+10 μg AGP-AF intranasallyin 20 μl volume. A tertiary immunization (3°) identical in compositionto the secondary immunization was administered 28 days after thesecondary immunization. Assays were conducted to detect cytotoxicT-lymphocyte activity at 16 days post secondary immunization (d16 post2°) and 8 days post tertiary immunization (d8 post 3°). Serum andmucosal antibody titers were assessed at 22 days post secondaryimmunization (d22 post 2°) and 21 days post tertiary immunization (d21post 3°). Antibody assays were conducted by standard ELISA methods.Cytotoxicity assays were conducted as described in TEST EXAMPLE 7.Results from this experiment are shown in Tables 23-26. TABLE 23Cytotoxic T-lymphocyte response of treated cells. % Cytotoxicity (d16,post 2°) E/T Material 50:1 25:1 12.5:1 6.25:1 B25 36 20 13 9 B15 13 5 44 B19 26 20 11 9 B4 28 17 9 7 B3 43 26 17 11 B5 43 30 20 11 B22 33 21 158 Vehicle 3 2 0 0 Normal 3 3 0 0 Cells

[0373] TABLE 24 Cytotoxic T-lymphocyte response of treated cells. %Cytotoxicity (d8, post 3°) E/T Material 50:1 25:1 12.5:1 6.25:1 B25 3019 13 8 B15 56 42 25 16 B19 71 54 33 24 B4 23 15 9 5 B3 54 45 32 20 B544 30 19 12 B22 22 13 7 5 Vehicle 5 2 1 1 Normal 7 5 3 3 Cells

[0374] TABLE 25 Anti-hepatitis antibody titers in treated mice. AntiHBsAg Titer⁻¹* Material IgG₁ IgG_(2a) IgA B25  256K# 500K 3,200 B15 256K500K 6,400 B19 500K  64K 1,600 B4 500K 1000K  6,400 B3 1000K  500K 6,400B5 256K 500K 3,200 B22 256K  64K 1,600 Vehicle  <2K  <2K <200

[0375] TABLE 26 Anti-hepatitis antibody titers in treated mice. AntiHBsAg Titer⁻¹* Material IgG₁ IgG_(2a) IgA B25  1000K# 1000K 25,600 B152000K 2000K 25,600 B19 2000K  500K 12,800 B4 1000K 2000K 25,600 B3 1000K1000K 25,600 B5  500K 1000K 12,800 B22  500K  500K 12,800 Vehicle   <2K  <2K <200

[0376] Groups of BALB/c mice were immunized with 2.5 μg HBsAg +10 μgAGP-AF intranasally and boosted intranasally with 7.5 μg HBsAg +10 μgAGP-AF 21 days later. Vaginal samples were collected 10 days after thebooster immunization and assayed for anti-HBsAg antibody. Results ofthis assay are shown in Table 27. TABLE 27 Vaginal Wash Anti-HBsAgTiter⁻¹ Material IgG IgA B25 100 800 B15 50 3200 B19 <50 400 B4 16006400 B3 800 1600 B5 1600 1600 B22 100 800 Vehicle <50 <50

[0377] The intranasal administration of HBsAg with the compounds of thesubject invention stimulated both a humoral and cellular immune responseto that antigen. Intranasal immunization with the antigen formulated inAGP-AF induced a cytotoxic T-lymphocyte response (Table 23-24) andantigen specific humoral (Table 25 and 26) and mucosal (Table 27) immuneresponses.

TEST EXAMPLE 10 Generation of a Protective Immune Response to Influenza

[0378] Mice immunized intranasally with FLUSHIELD influenza vaccinecontaining hemagglutinin antigen and the AGPs of the subject inventionproduced both IgG and IgA, which were recovered in vaginal washes.Immunized mice were also protected from subsequent influenza challenge.

[0379] ICR mice were immunized three times at 21 day intervalsintranasally with FLUSHIELD influenza vaccine (Wyeth-Lederle) containing0.3 μg hemagglutinin antigen (HA)+10 μg AGP-AF or recombinant E. coliheat labile enterotoxin (LT). AGP-AF was prepared as in TEST EXAMPLE 8.LT was solubilized in saline at 1 μg/ml. Vaginal washes were collected14 days after the second and third immunization. Serum samples werecollected 14 days after the third immunization. Mice were challengedwith 10 LD₅₀ (lethal dose 50) of infectious influenza A/HK/68thirty-five days after the final immunization and monitored formortality. Tables 28 and 29 show the results of assays conducted bystandard ELISA methods to detect anti-influenza antibody titers invaginal washes and sera. TABLE 28 Vaginal Wash Samples IgA IgG PercentMaterial Secondary Tertiary Secondary Tertiary Protection Nonimmune <20<20 <20 <20 22 Vehicle 80 160 160 160 50 B25 1280 1280 640 2560 100 B19320 5120 1280 1280 70 B3 1280 2560 1280 1280 100 B22 640 2560 320 640 75LT 2560 2560 2560 640 100

[0380] TABLE 29 Serum Titers Percent Material Total IgG IgG₁ IgG_(2a)IgG_(2b) Protection Nonimmune <400 <400 <400 <400 22 Vehicle 102,400256,000 12,800 102,400 50 B25 ≧819,200 102,400 819,200 ≧819,200 100 B19819,200 51,200 102,400 819,200 70 B3 ≧819,200 51,200 819,200 ≧819,200100 B22 819,200 51,200 102,400 819,200 75 LT ≧819,200 ≧819,200 ≧819,200≧819,200 100

[0381] These data demonstrate that AGPs in AF when administeredintranasally act as a mucosal adjuvants causing the production of IgA atmucosal sites. Increased protection is also induced against an upperrespiratory pathogen that invades through the mucosa.

[0382] A second experiment was performed similar to that described aboveusing BABL/c mice and 3 vaccinations at 2-week intervals. In addition toAGP formulations, an aqueous formulation of MPL® was also tested. Theaqueous AGP and MPL® formulations were prepared containing 1 mg/ml AGPor 3-O-deacylated monophosphoryl lipid A in 0.2% triethanolamine/water.Mice were challenged with approximately 2 LD₅₀ of infectious influenzaA/HK/68 twenty-eight days after the final immunization and monitored formortality. Table 30 shows the results of assays conducted by standardELISA methods to detect anti-influenza antibody titers in vaginal andtracheal washes and sera. TABLE 30 Mucosal IgA Serum IgG ResponsePercent Material Titers TW VW Protection Vehicle 5.6 <4.6 <4.6 0Flu/Vehicle 10.6 <5.0 6.6 0 MPL ® 17.1 9.3 9.6 63 B34 8.4 <4.6 <4.6 0B15 19.1 11.1 10.9 100

[0383] These data demonstrate that B 15 and MPL® when administeredintranasally act as mucosal adjuvants causing the production of IgA attwo mucosal sites. Increased protection is also induced against an upperrespiratory pathogen that invades through the mucosa.

TEST EXAMPLE 11 Generation of Immune Responses from Stable EmulsionFormulations

[0384] The AGP compounds of the subject invention stimulated bothhumoral and cytotoxic T-lymphocyte responses when formulated in a stableemulsion (SE). AGPs were tested at 25 μg dose levels to adjuvantizeHepatitis B surface antigen (HBsAg) for the induction of CTL andantibody responses. BALB/c mice were immunized subcutaneously with 2.0μg HBsAg plus 25 μg of AGP/SE on day 0 and day 21. The CTL assay wasconducted as in TEST EXAMPLE 7. The AGPs were formulated in a stableemulsion (SE) and the compositions were designated AGP-SE. Methods forpreparing the stable emulsion containing 10% v/v squalene, 0.091% w/vPLURONIC-F68 block copolymer, 1.909% w/v egg phosphatidyl choline, 1.8%v/v glycerol, 0.05% w/v α tocopherol, 10% ammonium phosphate buffer and78.2% v/v Water for Injection should be readily apparent to one skilledin the art. The emulsion was homogenized to a particle size of >0.2 μm.Table 31 shows the AGPs of the subject invention induced a cytotoxicT-lymphocyte response to HBsAg. TABLE 31 Cytotoxic T-lymphocyte responseof treated cells. % Cytotoxicity E:T Material Day 50:1 25:1 12.5:16.25:1 B25 d17, post 1° 27 12 9 5 B19 74 48 34 24 B3 28 15 9 5 B22 42 2417 7 Vehicle-SE 32 16 9 6 B25 d16, post 2° 49 28 20 13 B19 73 62 42 31B3 81 47 32 22 B22 78 69 58 39 Vehicle-SE 38 23 14 8

[0385] The results of the antibody titer to HBsAg are shown on Table 32.Sera from bleeds taken on day 28 post 2° were titered on ELISA platescoated with either HBsAg or a 28 amino acid peptide (p72) which containsB-cell epitopes found in the S-antigen region, residues 110-137, of theHBsAg. TABLE 32 Anti-HBsAg titer of treated mice. Anti-HBsAg Titer⁻¹HBsAg p72-Peptide Material IgG₁ IgG_(2a) IgG₁ IgG_(2a) B25  2048K* 2048K128K   64K B19 1024K 1024K 64K 128K B3  512K 1024K 16K 128K B22 1024K1024K 128K  128K Vehicle SE 1024K   64K 64K   4K

[0386] AGP-SE treated mice displayed both humoral (Table 32) andcytotoxic T-lymphocyte (Table 31) responses to the hepatitis B surfaceantigen. Of interest, AGP-SE treated mice in serum displayed a vigorousIgG₂a specific antibody titer detected by both antigens, whereas thevehicle-SE induced only a modest IgG_(2a) response.

TEXT EXAMPLE 12 Stimulation of Serum Antibody Titers

[0387] The AGP compound B31 was evaluated for its ability to enhanceserum antibody titers to an influenza virus vaccine as set forth in TextExample 3. In brief, ICR mice (10/group) were administered vaccinescontaining 1 HAU of formalin-inactivated influenza A/HK/68 plus or minus25 μg RC-523 formulated in a 0.2% TEoA/water solution. The mice were,also, challenged with a lethal dose of infectious influenza virus inorder to assess protection. The results of this experiment are presentedin Table 33. TABLE 33 Anti-influenza serum titers Material IgG IgG1IgG2a IgG2b Protection Nonimmune 200 50 50 100 0 Vehicle 200 200 50 20025 B31 3200 1600 400 1600 70

TEXT EXAMPLE 13 Inducible Nitric Oxide Synthetic Activity

[0388] Screening of respective AGP compounds of this invention includedevaluation of inducible nitric oxide synthetase or iNOS activity (NOSED₅₀), which correlates with macrophage activation, and can thus beviewed as a measure of immune stimulation. For this assay, mouseperitoneal exudates cells were harvested and the adherent cellpopulation isolated. The adherent cells were exposed to varyingconcentrations of soluble AGP compounds and the resulting induction andsecretion of nitrite measured. The NOS ED₅₀ value represents aconcentration of AGP required to stimulate half the maximum amount ofnitrite release and corresponds to the concentration required tostimulate macrophages.

[0389] The AGP compounds were also evaluated for their tendency toinduce a fever response in rabbits. Each compound was formulated in 10%(v/v) ethanol/water solution at 100 mg/ml, then diluted with D₅W to thedesired concentration. The material was injected at 3 ml/kg body weightinto 3 rabbits. The rise in core temperature of the rabbits wasrecorded. A compound inducing a cumulative rise of greater than or equalto 1.5 degree in the three rabbits is considered pyrogenic.

[0390] The results of these experiments are presented in Table 34. TABLE34 NOS PYROGENICITY ED₅₀ Total Rise ° C., 3 rabbits (nanograms/ml) 2.510 MPD # Exp. 1 Exp 2 Exp 3 ug/kg ug/kg ug/kg B1 150 0 0.1 B2 9 0.9 3.62.5-5 B3 4 0 4.2 B4 5 0.1 3.4 B5 5 3 0.1 4.1 B6 1.8 2.1 — B7 21 0 4 B8≧3000 3.6 — B9 16 0 3.1 B10 4 0 5.8 B11 3 4.2 —   0.3-0.6 B12 0.9 2.5 —B13 0.1 3 — B14 0.25 2.1 — B15 0.06 4.2 3.1 <0.06 B16 0.46 1.8 — B1732.5 2.1 3.4 B18 ≧3000 4.3 — B19 100 0 0.3 B20 0.5 1.5 0.3 4.6 B21 8 0.52 B22 51 1.7 — B23 159 0.3 0.3 B24 20 17 0.9 2.4 2.5-5 B25 0.3 0.5 0.64.2 B26 67 0.2 1.7 5 B27 1.65 1.8 3.9 B28 0.3 4.2 — B29 ≧10,000 0.2 0.7B30 4.3 — B31 ≧10,000 0.5 1.6 B32 B34 ≧10,000 3.5 2.8 B35 86 3.2 — B361.8 3.4 — B37 1.1 2.2 — B38 ≧3000 3.6 — B39 ≧3000 3.2 — B40 ≧3000 3.8 —B41 6.3 — B42 5.2 — B43 380 3.8 —

TEXT EXAMPLE 14 Clinical Efficacy Data

[0391] This Example discloses primary efficacy results of a randomized,controlled study comparing the efficacy and safety of the AGP designatedRC-210-04 (B 19 in Table 1 and Example 20 herein above; chemical name2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranosideTriethylammonium Salt)). Hepatitis B surface antigen (AgB) wasadministered to healthy adults, who are not immune to Hepatitis B virus(HBV), either alone or in conjunction with RC-210-04. After screening,subjects were randomized to one of two treatment arms and received threeintramuscular (IM) injections of either AgB/RC-210-04 or AgB on Days 0,30, and 180. Subjects were also seen by the study physician(s) on Days60, 90, and 210 to evaluate safety and efficacy parameters. The primaryefficacy objective of the study was to evaluate the number of subjectswho became seroprotected (anti-HBsAg titer of ≧10 MIU/mL) at Day 90 withAgB/RC-210-04 compared to AgB.

[0392] Patient Demographics

[0393] Subject disposition data are summarized below for theintent-to-treat (ITT) population, defined as all subjects who wererandomized. Summaries and statistical analyses of the demographic andefficacy data are presented for the efficacy evaluable (EE) population,defined as all subjects who were randomized and received both the Day 0and Day 30 injections.

[0394] A total of 341 subjects were randomized at two sites (i.e., theITT population). Table 35 summarizes the randomization of subjects ateach center, the total number of subjects in the EE population, and thenumber of subjects in the EE population with data at the Day 0, Day 30,Day 60 and Day 90 visits. TABLE 35 Subject Enrollment and Disposition byInvestigator/Center # Subjects in Investigator # Subjects in Efficacy #Subjects in Efficacy Population with Data at Visit Center No. ITTPopulation Population Day 0 Day 30 Day 60 Day 90 Dupont 101 181 155 155155 152 148 Altclas 102 160 117 117 117 112 102 Total: 341 272 272 272264 250

[0395] Table 36 summarizes the disposition of subjects and the reasonsfor discontinuation of treatment according to treatment group. A totalof 272 of the 341 randomized subjects received both the Day 0 and Day 30doses of study medication (EE Population). The treatment groups werecomparable with respect to the number (%) of subjects in the EEpopulation (p=0.332).

[0396] A total of 78/341 subjects (23%) discontinued participation inthe study prior to the Day 90 visit (Table 36). The treatment groupswere comparable with respect to the number (%) of subjects whodiscontinued participation in the study (p=0.187). The most frequentreason for discontinuation was lost to follow-up (70/341 subjects[21%]). There was no apparent difference between the treatment groups interms of withdrawal rates or reason for withdrawal TABLE 36 SubjectDisposition and Reason for Discontinuation: ITT Population AgBAgB/RC-210-04 Randomized 171 170 Number of vaccinations 0 22 34 1 9 4 2117 106 3 23 26 Efficacy Evaluable 140 (81.9%) 132 (77.6%)Population^(a) Discontinued 34 (19.9%) 44 (25.9%) Reason fordiscontinuation Lost to follow-up 30 (17.5%) 42 (24.7%) Withdrew consent2 (1.2%) Investigator decision 1 (0.6%) 2 (1.2%) Unknown 1 (0.6%)

[0397] A total of 263/341 subjects (77%) completed the Day 90 visit. Thepercentages of subjects who completed the Day 90 visit were similar forthe two treatment groups. The percentages of subjects who completed eachstudy visit are shown in Table 37. TABLE 37 Number (%) of Subjects WhoCompleted Study Visits: ITT Population AgB AgB/RC-210-04 Visit Completed(N = 171) (N = 170) Day 0 148 (86.6%) 136 (80.0%) Day 30 141 (82.5%) 132(77.6%) Day 60 137 (80.1%) 128 (75.3%) Day 90 130 (76.0%) 121 (71.2%)

[0398] Table 38 summarizes the demographic characteristics for the EEpopulation. The study population was predominantly Caucasian, with aroughly equal division between male and female subjects. The mean agewas about 27 years, but subjects in the AgB/RC-210-04 treatment groupwere significantly younger than those in the AgB treatment group(p=0.016). 50% of the subjects in the AgB/RC-210-04 treatment group wereless than 25 years old, compared to 32% of subjects in the AgB treatmentgroup (p=0.003). There were no other significant differences between thetreatment groups with respect to the demographic variables summarized inTable 38 (p≧0.222). TABLE 38 Demographic Characteristics: EE PopulationAgB AgB/RC-210-04 Characteristic (N = 140) (N = 132) p-value Age (yrs) n140 132 mean ± SD 28.2 ± 6.0  26.4 ± 6.2  0.016^(a) median 28 25 range19-40 17-41 <25 45 (32.1%) 66 (50.0%) 0.003^(b) ≧35 28 (20.0%) 19(14.4%) 0.222^(b) Weight (kg) n 140 132 mean ± SD 70.0 ± 17.3 68.4 ±13.6 0.398^(a) median 68 67 Gender Male 68 (48.6%) 67 (50.8%) 0.719^(b)Female 72 (51.4%) 65 (49.2%) Race Caucasian 126 (90.0%) 120 (90.9%)0.799^(b) Non-Caucasian 14 (10.0%) 12 (9.1%)

[0399] Analysis of Efficacy

[0400] The primary efficacy endpoint was the number (%) of subjectsachieving seroprotection at the Day 90 visit, defined as an anti-HBsAgtiter of ≧10 MIU/mL. Secondary efficacy endpoints were: (1) the number(%) of subjects achieving seroprotection at the Day 30 and Day 60visits; (2) the number (%) of subjects achieving seroconversion (definedas an anti-HBsAg titer of ≧1 MIU/mL) at the Day 30, Day 60 and Day 90visits; and (3) the log-transformed anti-HBsAg titer levels at Days 30,Day 60, and Day 90.

[0401] Primary Efficacy Analysis: Seroprotection at Day 90

[0402] In the EE population, 126 of 132 (95.5%) of the subjects in theAgB/RC-210-04 treatment group achieved seroprotection by the Day 90visit, compared to 115 of 140 (82.1%) of the subjects in the AgBtreatment group. The difference in percentage of subjects achievingseroprotection was statistically significant (p=0.001). FIG. 1summarizes the percentages of subjects in the EE population achievingseroprotection at the Day 90 visit for each of the two investigatorsites, as well as for the two sites combined. These data showed thatthere were significantly more subjects seroprotected after twoimmunizations with AgB/RC-210-04 than with AgB alone.

[0403] Secondary Efficacy Analysis: Seroprotection at Day 30 and Day 60

[0404] In the AgB/RC-210-04 treatment group, 29 of the 132 subjects(22.0%) had achieved seroprotection by the Day 30 visit, compared to 9of the 140 subjects (6.4%) in the AgB treatment group. The differencebetween treatment groups in the rate of seroprotection by Day 30 wasstatistically significant (p<0.001). By the Day 60 visit, 123 of the 132subjects (93.2%) in the AgB/RC-210-04 treatment group had achievedseroprotection, compared to 99 of the 140 subjects (70.7%) in the AgBtreatment group. The between treatment group difference in the rate ofseroprotection by the Day 60 visit was statistically significant(p<0.001). FIG. 2 summarizes the percentages of subjects in the twotreatment groups who achieved seroprotection at each of the Day 30, 60,and 90 visits.

[0405] Secondary Efficacy Analysis: Seroconversion at Days 30, 60, and90

[0406] In the AgB/RC-210-04 treatment group, 72 of 132 subjects (54.6%)had achieved seroconversion at the Day 30 visit, compared to 30 of 140subjects (21.4%) in the AgB treatment group. The difference betweentreatment groups in rate of seroconversion at the Day 30 visit wasstatistically significant (p<0.001). At the Day 60 visit, 128 of the 132subjects (97.0%) in the AgB/RC-210-04 treatment group had achievedseroconversion, compared to 119 of 140 subjects (85.0%) in the AgBtreatment group. The rate of seroconversion at the Day 60 visit differedsignificantly between the two treatment groups (p<0.001). At the Day 90visit, 126 of the 132 subjects (95.5%) in the AgB/RC-210-04 treatmentgroup had achieved seroconversion, while 133 of the 140 subjects (95.0%)in the AgB treatment group had achieved seroconversion. There was nostatistically significant difference between the treatment groups inrate of seroconversion at the Day 90 visit (p=0.861). FIG. 3 summarizesthe percentages of subjects in the two treatment groups who achievedseroconversion at each of the Day 30, 60, and 90 visits.

[0407] Secondary Efficacy Analysis: Log-Transformed anti HBsAg TiterLevels EE Population at Days 30, Day 60, and Day 90

[0408] Table 39 summarizes the log-transformed anti-HBsAg titer levelsin each treatment group, at each of the Day 30, Day 60, and Day 90visits. Table 39 also shows the geometric mean of the anti-HBsAg titerlevels in each dose at each visit (as the antilog of the mean of thelog-transformed titer levels). Significant differences in meanlog-transformed titer levels were found between the treatment groups ateach of the Day 30, Day 60, and Day 90 visits (p<0.001, each visit).TABLE 39 Log-Transformed Anti-HBsAg liter Levels, Days 30, 60, 90: EEPopulation AgB AgB/RC-210-04 Visit (N = 140) (N = 132) p-value Day 30 n139 132 mean ± SD −0.66 ± 0.98  0.12 ± 1.21 <0.001^(a) antilog(mean)0.22 1.33 Day 60 n 137 128 mean ± SD 1.37 ± 1.16 2.23 ± 0.68 <0.001^(a)antilog(mean) 23.37 170.93 Day 90 n 137 130 mean ± SD 1.86 ± 0.86 2.48 ±0.61 <0.001^(a) antilog(mean) 71.66 302.62

[0409]FIG. 4 shows the estimated distributions of anti-HBsAg titerlevels in both treatment groups at each of Days 30, 60, and 90. Theestimated distributions were obtained by applying a nonparametricdensity estimator to the observed titer levels for all EE populationpatients for whom a positive titer level was obtained.

[0410] Results of all three secondary efficacy endpoints (seroprotectionat Days 30 and 60, seroconversion rates, and geometric mean titers)supported the results of the primary endpoint. Significantly highergeometric mean titers in the AgB/RC-210-04 group suggest thatpost-vaccination titer level correlated with duration of seroprotection.

[0411] In total, the data presented in Test Example 14 demonstrate thataddition of RC-210-04 to AgB resulted in an adjuvanted vaccine thatillicited faster and greater antibody responses in healthy adults toHepatitis B surface antigen. AgB/RC-210-04 was efficacious for inducingearly seroprotection to Hepatitis B virus after two immunizations.

[0412] It is understood that the foregoing examples are merelyillustrative of the present invention. Certain modifications of thecompositions and/or methods employed may be made and still achieve theobjectives of the invention. Such modifications are contemplated aswithin the scope of the claimed invention.

REFERENCES

[0413] Bulusu, M. A. R. C., Waldstatten, P., Hildebrandt, J., Schütze,E. and G. Schulz (1992) Cyclic Analogues of Lipid A: Synthesis andBiological Activities, J. Med. Chem. 35: 3463-3469.

[0414] Ikeda, K., Asahara, T. and K. Achiwa (1993) Synthesis ofBiologically Active N-acylated L-serine-ContainingGlucosaminide-4-Phosphate Derivatives of Lipid A, Chem. Pharm. Bull.41(10): 1879-1881.

[0415] Miyajima, K., Ikeda, K. and K. Achiwa (1996) Lipid A and RelatedCompounds XXXI. Synthesis of Biologically Active N-AcylatedL-Serine-Containing D-Glucosaminide 4-Phosphate Derivatives of Lipid A,Chem. Pharm. Bull. 44(12): 2268-2273.

[0416] Shimizu, T., Akiyama, S., Masuzawa, T., Yanagihara, Y., Nakamoto,S., Takahashi, T., Ikeda, K. and K. Achiwa (1985) Antitumor Activity andBiological Effects of Chemically Synthesized Monosaccharide Analogues ofLipid A in Mice. Chem. Pharm. Bull. 33(10): 4621-4624.

[0417] Shimizu, T., Sugiyama, K., Iwamoto, Y., Yanagihara, Y., Asahara,T., Ikeda, K. and K. Achiwa (1994) Biological Activities of ChemicallySynthesized N-acylated Serine-linked Lipid A Analog in Mice, Int. J.Immunopharmac., 16(8): 659-665.

[0418] Shimizu, T., Iida, K., Iwamoto, Y., Yanagihara, Y., Ryoyama, K.,Asahara, T., Ikeda, K. and K. Achiwa (1995) Biological Activities andAntitumor Effects of Synthetic Lipid A Analogs Linked N-Acylated Serine,Int. J. Immunopharmac., 17(5): 425-431.

What is claimed is:
 1. An immunoeffector compound having the followingstructure:

wherein, X is selected from the group consisting of O and S at the axialor equitorial position; Y is selected from the group consisting of O andNH; n, m, p and q are integers from 0 to 6; R₁, R₂ and R₃ are the sameor different and are normal fatty acyl residues having from 1 to about20 carbon atoms and where one of R₁, R₂ or R₃ is optionally hydrogen; R₄and R₅ are the same or different and are selected from the groupconsisting of H and methyl; R₆ and R₇ are the same or different and areselected from the group consisting of H, hydroxy, alkoxy, phosphono,phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl andcarboxy, and esters and amides thereof; and R₈ and R₉ are the same ordifferent and are selected from the group consisting of phosphono and H,and at least one of R₈ and R₉ is phosphono.
 2. The compound of claim 1,wherein R₆ is carboxy.
 3. The compound of claim 2, wherein X is O; Y isO; n, m, p and q are 0; R₁, R₂ and R₃ are normal fatty acyl residueshaving 10 carbon atoms; R₄, R₅ and R₇ are H; R₈ is phosphono; R₉ is H;R₁, R₂ and R₃ are each attached to a stereogenic center having an Rconfiguration; and R₅ is attached to a stereogenic center having an Sconfiguration.
 4. The compound of claim 2, wherein X is O; Y is O; n, m,p and q are 0; R₁, R₂ and R₃ are normal fatty acyl residues having 12carbon atoms; R₄, R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ andR₃ are each attached to a stereogenic center having an R configuration;and R₅ is attached to a stereogenic center having an S configuration. 5.The compound of claim 2, wherein X is O; Y is O; n, m, p and q are 0;R₁, R₂ and R₃ are normal fatty acyl residues having 10 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an R configuration.
 6. Thecompound of claim 2, wherein X is O; Y is O; n, m, p and q are 0; R₁, R₂and R₃ are normal fatty acyl residues having 8 carbon atoms; R₄, R₅ andR₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are each attached to astereogenic center having an R configuration; and R₅ is attached to astereogenic center having an S configuration.
 7. The compound of claim1, wherein R₆ is H.
 8. The compound of claim 7, wherein X is O; Y is O;n is 2; m, p and q are 0; R₁, R₂ and R₃ are normal fatty acyl residueshaving 14 carbon atoms; R₄, R₅ and R₇ are H; R₈ is phosphono; R₉ is H;and R₁, R₂ and R₃ are each attached to a stereogenic center having an Rconfiguration.
 9. The compound of claim 7, wherein X is O; Y is O; n is1, m and p are 0; q is 1; R₁, R₂ and R₃ are normal fatty acyl residueshaving 10 carbon atoms; R₄ and R₅ are H; R₇ is carboxy; R₈ is phosphono;R₉ is H; and R₁, R₂ and R₃ are each attached to a stereogenic centerhaving an R configuration.
 10. The compound of claim 7, wherein X is O;Y is O; m, n, p and q are 0; R₁, R₂ and R₃ are normal fatty acylresidues having 14 carbon atoms; R₄, R₅ and R₇ are H; R₉ is phosphono;R₉ is H; and R₁, R₂ and R₃ are each attached to a stereogenic centerhaving an R configuration.
 11. The compound of claim 7, wherein X is O;Y is O; m, n, p and q are 0; R₁, R₂ and R₃ are normal fatty acylresidues having 10 carbon atoms; R₄, R₅ and R₇ are H; R₈ is phosphono;R₉ is H; and R₁, R₂ and R₃ are each attached to a stereogenic centerhaving an R configuration.
 12. The compound of claim 7, wherein X is O;Y is O; m, p and q are 0; n is 1; R₁, R₂ and R₃ are normal fatty acylresidues having 14 carbons; R₄, R₅ and R₇ are H; R₈ is phosphono; R₉ isH; and R₁, R₂ and R₃ are each attached to a stereogenic center having anR configuration.
 13. The compound of claim 1, wherein R₆ is hydroxy. 14.The compound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is1; R₁, R₂ and R₃ are normal fatty acyl residues having 12 carbon atoms;R₄ and R₅ are H; R₇ is H; R₈ is phosphono; and R₉ is H; R₁, R₂ and R₃are each attached to a stereogenic center having an R configuration; andR₅ is attached to a stereogenic center having an S configuration. 15.The compound of claim 13, wherein X is O; Y is O; m and q are 0; n and pare 1; R₁, R₂ and R₃ are normal fatty acyl residues having 10 carbonatoms; R₄, R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ areeach attached to a stereogenic center having an R configuration; and R₅is attached to a stereogenic center having an S configuration.
 16. Thecompound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is 2;R₁, R₂ and R₃ are normal fatty acyl residues having 10 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an S configuration.
 17. Thecompound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is 1;R₁, R₂ and R₃ are normal fatty acyl residues having 14 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an R configuration.
 18. Thecompound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is 1;R₁, R₂ and R₃ are normal fatty acyl residues having 14 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an S configuration.
 19. Thecompound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is 1;R₁, R₂ and R₃ are normal fatty acyl residues having 11 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an S configuration.
 20. Thecompound of claim 13, wherein X is O; Y is O; m, n and q are 0; p is 1;R₁, R₂ and R₃ are normal fatty acyl residues having 10 carbon atoms; R₄,R₅ and R₇ are H; R₈ is phosphono; R₉ is H; R₁, R₂ and R₃ are eachattached to a stereogenic center having an R configuration; and R₅ isattached to a stereogenic center having an S configuration.
 21. Thecompound of claim 1, wherein X is O; Y is O; m, n, p and q are 0; R₁, R₂and R₃ are normal fatty acyl residues having 10 carbon atoms; R₄ and R₅are H; R₆ is amino carbonyl; R₇ is H; R₈ is phosphono; and R₉ is H; R₁,R₂ and R₃ are each attached to a stereogenic center having an Rconfiguration; and R₅ is attached to a stereogenic center having an Sconfiguration.
 22. The compound of claim 1, wherein R₁ is hydrogen. 23.The compound of claim 1, wherein R₂ is hydrogen.
 24. The compound ofclaim 1, wherein R₃ is hydrogen.
 25. A method for enhancing the immuneresponse of a mammal comprising administering to the mammal an effectiveamount of a compound of claim
 1. 26. An immunogenic compositioncomprising a compound of claim 1, an antigen and a suitable carrier. 27.A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 28. The composition of claim 27,wherein said pharmaceutically acceptable carrier is an aqueouscomposition comprising water and one or more surfactants selected fromthe group consisting of glycodeoxycholate, deoxycholate, sphingomyelin,sphingosine, phosphatidylcholine,1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine,L-α-Phosphatidylethanolamine, and1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, or a mixture thereof. 29.The composition of claim 28, wherein said one or more surfactant is1,2-Dipalmitoyl-sn-glycero-3-phosphocholine.
 30. The composition ofclaim 28, wherein the molar ratio of said compound to surfactant is fromabout 10:1 to about 1:25.
 31. The composition of claim 28, wherein themolar ratio of said compound to surfactant is from about 4:1 to about1:9.
 32. The composition of claim 27, wherein said carrier is a stableemulsion comprising a metabolizable oil, one or more surfactants, anantioxidant and a component to make the emulsion isotonic.
 33. Thecomposition of claim 32, wherein said stable emulsion comprises 1-10%v/v squalene, 0.9% w/v PLURONIC-F68 block co-polymer, 1.9% w/v eggphosphatidyl choline, 1.75% v/v glycerol and 0.05% w/v α tocopherol. 34.The composition of claim 27 wherein said carrier is a suspensioncomprising aluminum hydroxide, calcium hydroxide, calcium phosphate ortyrosine adsorbate.
 35. The composition of claim 27 wherein said carrieris an aqueous solution or aqueous micellar dispersion comprisingtriethylamine or triethanolamine.
 36. The composition of claim 27wherein said carrier comprises microspheres or microparticles, and thecompound of claim 1 is within the matrix of the microspheres ormicroparticles or adsorbed thereon.
 37. A composition comprising acompound of claim 1 and one or more polypeptide.
 38. The composition ofclaim 37 wherein said compound is a2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranoside.39. The composition of claim 38 wherein said polypeptide is thehepatitis B surface antigen.
 40. A composition comprising a compound ofclaim 1 and one or more polynucleotide.
 41. The composition of claim 40wherein said polynucleotide encodes a polypeptide.
 42. The compositionof claim 38 wherein said compound is a2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl2-Deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradecanoylamino]-β-D-glucopyranoside.43. A method for illiciting an immune response in a mammal, comprisingthe step of administering a composition of claim
 37. 44. The method ofclaim 43 wherein said immune response is immunoprotective.
 45. Themethod of claim 43 wherein said mammal is a human.
 46. A method forilliciting an immune response in a mammal, comprising the step ofadministering a composition of claim
 40. 47. The method of claim 46wherein said immune response is immunoprotective.
 48. The method ofclaim 46 wherein said mammal is a human.